Gene therapy for ocular disorders

ABSTRACT

Compositions and methods are provided for treating Leber congenital amaurosis (LCA) in a subject. In one aspect, a recombinant adeno-associated viral vector is provided which includes a nucleic acid molecule comprising a sequence encoding Lebercilin. In another aspect, Lebercilin has an amino acid sequence of SEQ ID NO: 1. In yet another aspect, the nucleic acid molecule has a sequence of SEQ ID NO: 3 or a variant thereof. In desired embodiments, the subject is human, cat, dog, sheep, or non-human primate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/489,770, filed Aug. 29, 2019, which is a national stage entry ofPCT/US2018/020470, filed Mar. 1, 2018, which claims benefit of U.S.Patent Application No. 62/465,649, filed Mar. 1, 208, and U.S. PatentApplication No. 62/469,642, filed Mar. 10, 2017, which are herebyincorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“16-7696.US.C1.xml”, is XX bytes, and was created on Dec. 5, 2022. Thecontents of the electronic sequence listing, including the sequences andtext therein, are incorporated herein by reference their entirety.

BACKGROUND OF THE INVENTION

One of the most severe groups of inherited blinding diseases is Lebercongenital amaurosis (LCA; OMIM 204000). LCA is rare, occurring in1:50,000 individuals, is usually inherited in an autosomal recessivefashion, and can be caused by mutations in any of at least 22 differentgenes (sph.uth.edu/RetNet/sum-dis.htm). Clinical features includeseverely abnormal vision (visual acuity, reduced visual fields) ininfancy or early childhood, nystagmus, and progressive loss of the poorvision that exists early in life. Clinical testing reveals extinguishedscotopic and photopic electroretinogram (ERG) responses, amauroticpupils, reduced light sensitivity, and pigmentary changes in the retina.There is currently no approved treatment for LCA.

A form of LCA that is caused by mutations in the retinal pigmentepithelium 65 kDa protein-encoding gene, RPE65 (Redmond T M, Yu S, LeeE, Bok D, Hamasaki D, Chen N, et al. Rpe65 is necessary for productionof 11-cis-vitamin A in the retinal visual cycle. Nat Genet (1998)20(4):344-51; Redmond T, Hamel C. Genetic analysis of RPE65: from humandisease to mouse model. Methods in Enzymol (2000) 317:705-24, both ofwhich are incorporated herein by reference), has received a great dealof attention in recent years as it has been the target of multiple geneaugmentation therapy clinical trials. Using adeno-associated virus (AAV)serotype 2, several groups have shown that delivery of the wildtype copyof the RPE65 cDNA to the retinal pigment epithelium (RPE) is safe, andthat this can reverse many of the deficits, including nyctalopia (3-9).A randomized, multi-center Phase 3 study testing AAV2-hRPE65v2 (orvoretigene neparvovec, sponsored by Spark Therapeutics, Philadelphia,Pa.), has shown that subretinal injection of this reagent leads toimprovements in light sensitivity, visual fields and even the ability tonavigate accurately and quickly using visual cues over a range ofluminance conditions (10, 11). The US Food and Drug Administration (FDA)granted drug approval for Voretigene neparvovec-rzyl on Dec. 19, 2017,making this one of the first approved gene therapy drugs in the USA. Theprogress in developing a treatment for LCA caused by RPE65 mutations,LCA2, paves the way for development of treatments for other forms ofearly onset retinal degeneration, most of which are caused by mutationsin photoreceptor-specific genes, not just RPE-specific genes.

One of the most severe forms of this already severe condition (LCA) iscaused by mutations in the photoreceptor-specific gene encodingLebercilin, LCA5 (12-20). LCA5 mutation is estimated to account for ˜2%of cases of LCA although it may be more prevalent in populations thatare genetically isolated (16). LCA5 mutations have also been identifiedas the cause of other early onset forms of retinal degeneration,including cone dystrophy and autosomal recessive retinitis pigmentosa(ARRP) (15, 16, 21).

Therefore, compositions useful for expressing Lebercilin in subjects inneed are needed.

SUMMARY OF THE INVENTION

The embodiments described herein are directed to compositions andmethods relating to an AAV gene therapy vector for delivering human LCA5to a subject in need thereof, following intravitreal or subretinaladministration of the vector resulting in long-term, perhaps 10 years ormore, of clinically meaningful correction of Leber congenital amaurosis(LCA).

In one aspect, a codon optimized, engineered nucleic acid sequenceencoding human Lebercilin is provided. In one embodiment, the codonoptimized nucleic acid sequence is a variant of SEQ ID NO: 3 or SEQ IDNo: 2. In another embodiment, the codon optimized nucleic acid sequenceis SEQ ID NO: 3. In another embodiment, the nucleic acid sequence iscodon optimized for expression in humans.

In another aspect, an expression cassette comprising a codon optimizednucleic acid sequence that encodes Lebercilin is provided. In oneembodiment, the expression cassette includes the nucleic acid sequenceof SEQ ID NO: 3 encoding human Lebercilin. In still other embodiments,the Lebercilin encoding sequence is positioned between 5′ and 3′ AAV ITRsequences.

In yet another aspect, a recombinant adeno-associated virus (rAAV)vector is provided. The rAAV compromises an AAV capsid, and a vectorgenome packaged therein. In one embodiment, the vector genome comprises:(a) an AAV 5′ inverted terminal repeat (ITR) sequence; (b) a promoter;(c) a coding sequence encoding a human Lebercilin; and (d) an AAV 3′ITR. In one embodiment, the rAAV vector further comprises expressioncontrol sequences that direct expression of the Lebercilin in a hostcell. In further embodiment, the Lebercilin sequence is the proteinsequence of SEQ ID NO: 1. In one embodiment, the vector genome is thesequence of nt 1-4379 of SEQ ID NO: 8. In another embodiment, the vectorgenome is the sequence of nt 1-4368 of SEQ ID NO: 9. In yet anotherembodiment, the LCA5 coding sequence in either of the identified vectorgenomes is swapped with another LCA5 coding sequence as describedherein.

In another aspect, an aqueous suspension suitable for administration toan LCA patient is provided. In one embodiment, the suspension comprisesan aqueous suspending liquid and about 1×10¹⁰ GC or viral particles toabout 1×10¹³ GC or viral particles per eye of a recombinantadeno-associated virus (rAAV) described herein useful as a therapeuticfor LCA.

In another aspect, a pharmaceutical composition comprises apharmaceutically acceptable carrier, diluent, excipient and/or adjuvantand the nucleic acid sequence, a plasmid, a vector, or a viral vector,such as the rAAV, described specifically herein.

In another aspect, a method for treating Leber Congenital Amaurosiscaused by a defect in the lebercilin gene (LCA5) and/or restoring visualfunction in a mammalian subject having LCA comprises delivering viaintravitreal, subretinal or intravascular injection to a subject in needthereof a recombinant AAV vector which encodes Lebercilin, as describedherein.

In another aspect, use of an AAV vector as described herein is providedin treating Leber Congenital Amaurosis caused by a defect in thelebercilin gene (LCA5) and/or restoring visual function in a mammaliansubject having LCA. The use includes delivering via intravitreal,subretinal or intravascular injection to a subject in need thereof arecombinant AAV vector which encodes Lebercilin, as described herein.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the transgene cassettes used to generateAAV7m8.CBA.hopt.LCA5 and AAV7m8.CBA.EGFP as described herein.

FIGS. 1B-1D provide an alignment of human nucleic acid sequence of LCA5(Native_LCA5) of SEQ ID NO: 2 and the codon optimized LCA5(Codon-optimized_LCA5) sequence of SEQ ID NO: 3.

FIGS. 1E-1F provide a plasmid map and a feature list of thepAAV.CMV.CBA.human codon-optimized Lebercilin vector. The nucleic acidsequence is reproduced in SEQ ID NO: 8.

FIGS. 1G-1H provide immunofluorescence analysis of eyes injected withAAV7m8-hopt-LCA5 at P5 and analyzed at P15 shows Lebercilinco-localizing with the base of tubulin-positive outer segments asdescribed in Examples 1, 2 and 4. After intravitreal injection (IVT,FIG. 1G) or subretinal injection (SR, FIG. 1L), Lebercilin wasdistributed throughout the retina, which was nearly devoid ofphotoreceptors at P95. In contrast, lebercilin is absent in untreatedP15 and P95 Lca5−/− retinas. SR, subretinal; IVT, intravitreal; (−)Untreated Lca5−/−. Cartoons are provided showing the intravitrealinjection scheme (FIG. 1G) and subretinal injection scheme (FIG. 1H).

FIG. 1I shows expression of Lebercilin in both wild type mouse andLca5−/− mice at P20 injected with the AAV.LCA5 vector subretinally.

FIGS. 2A-2I show normalized pupillary reflex amplitudes measured at 3months of age of AAV7m8.hopt-LCA5-injected mice compared to control(sham-injected) eyes of Lca5−/− mice treated at PN5 and PN15. Resultsare shown after (A) intravitreal and (B) subretinal injection or (C) inuntreated (−) control mice. (D) The relative pupillary reflex amplitudes(% of baseline pupil diameter) of the right eyes of animals in each ofthe sub-groups shown in (A-C) plus in wildtype (C57B16) positive (+)control mice are shown graphically (E). FIGS. 2F and 2G arerepresentations of the testing scheme used to generate the results shownin FIGS. 2A-2D. FIGS. 2H and 21 show a comparison of normalizedpupillary reflex amplitudes in experimental and control mice, shown inFIGS. 2A-2D, treated at PN5 vs PN15 via subretinal (SR, I) orintravitreal (IV, H) injection. *p<0.1; **p<0.05; ***p<0.01.

FIGS. 3A-3F show results of water maze test AAV7m8.hopt-LCA5-injectedvs. control (sham-injected) eyes of Lca5−/− mice treated at PN5 and PN15and measured at 3 months of age as described in Example 3. FIG. 3A is atable showing the statistical analysis result. FIG. 3B is anillustration of representative results of water maze test. FIG. 3D is abar graph showing days until first success in training of Lca5−/− miceat PN5 or PN15 treated at birth with the AAV7m8.LCA5 vectorintravitreally or subretinally at birth. Wild type mice were provided ascontrols. FIG. 3D is a line graph of success rates under various lightintensities (x-axis) of Lca5−/− mice at PN5 treated at birth with theAAV7m8.LCA5 vector intravitreally. FIG. 3E is a line graph of successrates under various light intensities (x-axis) of Lca5−/− mice at PN5treated at birth with the AAV7m8.LCA5 vector subretinally FIG. 3F is aline graph of success rates under various light intensities (x-axis) ofLca5−/− mice at PN15 treated at birth with the AAV7m8.LCA5 vectorintravitreally.

FIG. 4 is a graph providing results of representative histology afterAAV7m8.hopt-LCA5 delivery to the Lca5gt/gt retina at postnatal day(PN)5. The graph shows the number of rows in the outer nuclear layer(ONL) after IVT or SR treatment with AAV.LCA5 vs. sham-injection.

FIG. 5A provides result of immunofluorescence shows that rhodopsinpersists in the treated (but not control untreated) photoreceptorsthrough the 3 month timepoint. Occasional photoreceptor cells areeGFP-positive (region of injection identified through co-injection withAAV7m8.eGFP).

FIGS. 5B-5D provide Multi-electrode array (MEA) responses from Lca5gt/gtAAV7m8.hopt-LCA5-treated rods and cones are similar to those of wildtype (WT) retinas. (B) Response amplitude (difference in firing ratesbefore and after flash onset) vs. flash intensity data measured from perflash averaged traces (not shown). Responses from treated retinas are ashigh as 70% of the response from WT retina; there is minimal toabolished response from untreated retina. (C) Response amplitudes fromretinas of panel A for the 1st and 2nd intensity series runs(before/during and after brightest exposures at the end of the 1st run,during each intensity series run stimulation series intensity wasincreased from scotopic to the brightest photopic values in ˜0.5 logincrements) (D) Amplitudes of transient ON- (difference in firing ratesbefore and after flash onset), sustained ON- (difference before flashonset and offset) and OFF-responses (difference before and after flashoffset) as functions of the flash intensity for the 1st and 2ndintensity series runs. Shaded areas represent range of WT responses (4retinas, MEAN±STD), line-only traces give averaged WT responseamplitudes. Horizontal arrows illustrate rightward shift in sensitivitycaused by exposure to the brightest flashes at the end of the 1stintensity series run. Treated Lca5gt/gt s and WT retinas show similarresponse/intensity dependencies before and after bleaching, whileuntreated Lca5gt/gt. retina responses are flattened. Circles connectedby lines represent treated LCA5gt/gt. Triangles connected by linesrepresent control LCA5gt/gt.

FIGS. 6A-6E show that there is massive cell death during photoreceptordegeneration early in life in the untreated Lca5−/− retina (and delay ofthis degeneration after treatment with AAV.hopt.LCA5) as evidenced by(A) TUNEL assay (FIG. 6A; FIG. 6B-E, third row) and (B) rhodopsinimmunofluorescence analysis. (FIG. 6B-E, rows 1, 3 and 4).

FIGS. 7A-7D show that outer segments were present inAAV7m8.hopt.LCA5-treated Lca5−/− retinas but not in control Lca5−/−retinas. Transmission electron microscopic evaluation of the retinasinjected with AAV7m8.hopt.LCA5 reveals both rod and cone photoreceptorouter segments with stacked discs and connecting cilia in 3 month oldLca5−/−. No such structures were present in untreated Lca5−/− retinas.(A, B) PN80 after intravitreal injection at PN5 with AAV7m8p643 (codonoptimized Lebercilin), representative pictures; (A1-A3): 12K resolution,stitched pictures showing rows of photoreceptor cells, Rod cells (arrowheads), cone cells (arrows), many mitochondria of photoreceptor cells(asterisks); (B), 20K resolution; (B1) cross section of cilia showing9+0 structure of microtubule, membraneous disc of OS from Rod cell(arrows); (C) 30K resolution, basal body (arrow head); (C1) sagitalsection of connecting cilium; (D) 12K resolution, no photoreceptor cellsremained in ONL; (D1) pyknotic nuclei (arrow head) of a dying cell; (D2)floating remnants of dead cell organelles including rough ER (arrows).

FIG. 8 shows result of Pupillary Light Responses of one retina of anadult man with LCA5 (top line) having similar temporal characteristicsas those of an age-comparable normal-sighted man (bottom line). Theamplitude of constriction was reduced in the LCA5 patient compared tothe normal individual.

FIG. 9 is a table of the thickness of various retinal layers showingthat outer nuclear layer (ONL) thickness remains thicker over at least 3months in the treated (*) compared to the control retinas. There was notsuch a clear trend in the other retinal layers (outer plexiform layer,OPL; inner nuclear layer, INL, inner plexiform layer, IPL). For eachtime point, bars from left to right represent thicknesses of ONL, OPL,INL and IPL, respectively.

FIG. 10 provides results of Light-mediated changes in position ofphototransduction-specific molecules after injection withAAV7m8.hopt.LCA5.

FIGS. 11A-11B provide a plasmid map and a feature list of thepAAV.CMV.CBA.human native Lebercilin vector. The nucleic acid sequenceis reproduced in SEQ ID NO: 9.

FIGS. 12A-12F demonstrate cilia phenotype rescued in homozygous humanLCA5 p. (Q279*) iPSC-RPE after treatment with AAV7m8.hopt-LCA5. (A)Confocal images show hexagonal morphology of mature RPE cells along withthe immunofluorescently detectable RPE markers; ZO-1 and MITF. Phasecontrast (PC) images display the architecture of iPSC-RPE cultures ofRPE derived from both the normal-sighted person and LCA5 patient; (B)Quantitative real-time PCR (qRT-PCR) of LCA5 mRNA expression innormal-sighted control RPEs and LCA5 patient-derived RPEs. GAPDH is usedto normalize expression levels. (C) Western blot analyses showendogenous (*) Lebercilin protein in normal-sighted control cells thatare untreated (“-”) or treated with AAV7m8.eGFP (“G”). There is noendogenous lebercilin in untreated or AAV7m8.GFP-treated LCA5-affectedcells. There are robust levels of lebercilin after infection of cellsfrom both normal-sighted and LCA5 individuals with AAV7m8.LCA5 (“L”)Immunofluorescence analyses show presence of ArII3b-positive primarycilia in normal-sighted (D) and LCA5-derived (E) iPSC-RPE. Lebercilin ispresent in normal-sighted control and AAV7m8.LCA5-treated (but notAAV7m8.eGFP-treated) LCA5-affected cells. (F) Quantitative analysis ofnumber of cilia per cell in normal-sighted- vs. LCA5-iPSC-RPE showsrescue effect of cilia formation after treatment of LCA5-iPSC-RPE cellswith AAV7m8.LCA5 (but not with AAV7m8.eGFP).

DETAILED DESCRIPTION OF THE INVENTION

The methods and compositions described herein involve compositions andmethods for delivering a LCA5 nucleic acid sequence encoding Lebercilinprotein to subjects in need thereof for the treatment of Lebercongenital amaurosis (LCA). In one embodiment, such compositions involvecodon optimization of Lebercilin coding sequence. It is desirable toincrease the efficacy of the product, and thus, increase safety, since alower dose of reagent may be used. Also encompassed herein arecompositions which include the native Lebercilin coding sequences, asshown in SEQ ID NO: 2.

Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application. The definitions contained in this specification areprovided for clarity in describing the components and compositionsherein and are not intended to limit the claimed invention.

“Lebercilin” is encoded by the LCA5 gene on chromosome 6q14 and is aciliary protein that localizes to the connecting cilia of photoreceptorsand to the microtubules, centrioles and primary cilia of culturedmammalian cells.

Lebercilin is expressed widely throughout development and is found incilia of cultured cells as well as in the connecting cilium of maturephotoreceptor cells. The connecting cilium is a narrow structure betweenthe photoreceptor inner segment that harbors the biosynthetic machineryof the cell, and the outer segments that contain the opsin-driven visualcascade. The connecting cilium functions as a conduit, supporting thebi-directional trafficking of proteins and vesicles along ciliarymicrotubule tracks in a process known as intraflagellar transport (IFT).Applying quantitative affinity proteomics to a genetically engineeredLca5 mouse model, Boldt et al demonstrated that Lca5 loss of functiondisrupts IFT, thereby causing defects in photoreceptor outer segmentdevelopment and failed arrestin and opsin trafficking. The Lca5 null(Lca5gt/gt) mice lack cone and rod ERG responses and undergo an earlyand progressive retinal degeneration with only a single row of dispersednuclei (compared to 8-10 rows of contiguous cells in retinas of wildtypemice) present in the outer nuclear layer (ONL) by 2 months of age 19.

Mutations in LCA5 lead to an inherited form of retinal degenerationcalled Leber Congenital Amaurosis (LCA). The phenotype in affectedindividuals is limited to the eye and results in blindness. In 6families studied by den Hollander, 5 had homozygous nonsense andframeshift mutations and in one family the LCA5 transcript wascompletely absent. The nucleic acids encoding the Lebercilin cDNA or acodon-optimized version thereof are of the appropriate size to fit intoan adeno-associated virus (AAV) vector. See e.g., the sequences in FIGS.1A and 1E to 1F and FIG. 11A-11B. As described in the examples, below,using a rAAV-mediated gene augmentation strategy, it is shown thatretinal degeneration due to LCA5 mutations can be corrected. Suchtherapy is particularly advantageous if the wildtype or optimized copyof the gene is delivered early in life, e.g., in childhood or in earlypostnatal period. Further, this intravitreal or subretinaladministration used in one embodiment, provides the gene efficiently tothe target cells (e.g., photoreceptors).

The Lebercilin gene, LCA5, encodes Lebercilin, a 697 amino acid proteinthought to be involved in centrosomal or ciliary functions and minusend-directed microtubule transport. As used herein, the terms “LCA5” and“Lebercilin” are used interchangeably when referring to the codingsequence. The native nucleic acid sequences encoding human Lebercilinare reported at NCBI Reference Sequence NM_181714.3 (transcript variant1), NM_001122769.2 (transcript variant 2), XM_011535504.1 (transcriptvariant X1) and XM_005248665.4 (transcript variant X2), and reproducedhere in SEQ ID NO: 4, 5, 6 and 7, respectively. The native human aminoacid sequence of Lebercilin is reproduced here at SEQ ID NO: 1 (NCBIReference Sequence: NP 001116241.1 or NP 859065.2, as well asUniProtKB/Swiss-Prot ID: Q86VQ0-1). Mutations in the LCA5 gene areassociated with Leber's congenital amaurosis (LCA). In certainembodiments, the terms “LCA5” and “Lebercilin” are used interchangeably.

Leber congenital amaurosis (LCA) is an eye disorder that primarilyaffects the retina, which is the specialized tissue at the back of theeye that detects light and color. People with this disorder typicallyhave severe visual impairment beginning in infancy. The visualimpairment tends to be stable, although it may worsen very slowly overtime. Leber congenital amaurosis is also associated with other visionproblems, including an increased sensitivity to light (photophobia),involuntary movements of the eyes (nystagmus), and extremefarsightedness (hyperopia). The pupils, which usually expand andcontract in response to the amount of light entering the eye, do notreact normally to light. Instead, they expand and contract more slowlythan normal, or they may not respond to light at all. Additionally, theclear front covering of the eye (the cornea) may be cone-shaped andabnormally thin, a condition known as keratoconus. A specific behaviorcalled Franceschetti's oculo-digital sign is characteristic of Lebercongenital amaurosis. This sign consists of poking, pressing, andrubbing the eyes with a knuckle or finger. Researchers suspect that thisbehavior may contribute to deep-set eyes and keratoconus in affectedchildren. In rare cases, delayed development and intellectual disabilityhave been reported in people with the features of Leber congenitalamaurosis. However, researchers are uncertain whether these individualsactually have Leber congenital amaurosis or another syndrome withsimilar signs and symptoms. At least 13 types of Leber congenitalamaurosis have been described. The types are distinguished by theirgenetic cause, patterns of vision loss, and related eye abnormalities.

The term “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of nucleic acidsequences refers to the residues in the two sequences which are the samewhen aligned for correspondence. The length of sequence identitycomparison may be over the full-length of the genome, the full-length ofa gene coding sequence, or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired.

Percent identity may be readily determined for amino acid sequences overthe full-length of a protein, polypeptide, about 32 amino acids, about330 amino acids, or a peptide fragment thereof or the correspondingnucleic acid sequence coding sequences. A suitable amino acid fragmentmay be at least about 8 amino acids in length, and may be up to about700 amino acids. Generally, when referring to “identity”, “homology”, or“similarity” between two different sequences, “identity”, “homology” or“similarity” is determined in reference to “aligned” sequences.“Aligned” sequences or “alignments” refer to multiple nucleic acidsequences or protein (amino acids) sequences, often containingcorrections for missing or additional bases or amino acids as comparedto a reference sequence.

Identity may be determined by preparing an alignment of the sequencesand through the use of a variety of algorithms and/or computer programsknown in the art or commercially available [e.g., BLAST, ExPASy;ClustalO; FASTA; using, e.g., Needleman-Wunsch algorithm, Smith-Watermanalgorithm]. Alignments are performed using any of a variety of publiclyor commercially available Multiple Sequence Alignment Programs. Sequencealignment programs are available for amino acid sequences, e.g., the“Clustal Omega” “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”,and “Match-Box” programs. Generally, any of these programs are used atdefault settings, although one of skill in the art can alter thesesettings as needed. Alternatively, one of skill in the art can utilizeanother algorithm or computer program which provides at least the levelof identity or alignment as that provided by the referenced algorithmsand programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “Acomprehensive comparison of multiple sequence alignments”,27(13):2682-2690 (1999).

Multiple sequence alignment programs are also available for nucleic acidsequences. Examples of such programs include, “Clustal Omega” “ClustalW”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which areaccessible through Web Servers on the internet. Other sources for suchprograms are known to those of skill in the art. Alternatively, VectorNTI utilities are also used. There are also a number of algorithms knownin the art that can be used to measure nucleotide sequence identity,including those contained in the programs described above. As anotherexample, polynucleotide sequences can be compared using Fasta™, aprogram in GCG Version 6.1. Fasta™ provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences. For instance, percent sequence identity betweennucleic acid sequences can be determined using Fasta™ with its defaultparameters (a word size of 6 and the NOPAM factor for the scoringmatrix) as provided in GCG Version 6.1, herein incorporated byreference.

In one aspect, a codon optimized, engineered nucleic acid sequenceencoding human Lebercilin is provided. Preferably, the codon optimizedLebercilin coding sequence has less than about 80% identity, preferablyabout 75% identity or less to the full-length native Lebercilin codingsequence (FIGS. 1B-1D, SEQ ID NO: 2). In one embodiment, the codonoptimized Lebercilin coding sequence has about 74% identity with thenative Lebercilin coding sequence of SEQ ID NO: 2. In one embodiment,the codon optimized Lebercilin coding sequence is characterized byimproved translation rate as compared to native Lebercilin followingAAV-mediated delivery (e.g., rAAV). In one embodiment, the codonoptimized Lebercilin coding sequence shares less than about 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%,83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%,69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or less identity to the fulllength native Lebercilin coding sequence of SEQ ID NO: 2. In oneembodiment, the codon optimized nucleic acid sequence is a variant ofSEQ ID NO: 3. In another embodiment, the codon optimized nucleic acidsequence a sequence sharing about 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%,78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%,64%, 63%, 62%, 61% or greater identity with SEQ ID NO: 3. In oneembodiment, the codon optimized nucleic acid sequence is SEQ ID NO: 3.In another embodiment, the nucleic acid sequence is codon optimized forexpression in humans. In another embodiment, the lebercilin codingsequence is nt 1883 to nt 3976 of SEQ ID NO: 8. In other embodiments, adifferent Lebercilin coding sequence is selected.

Codon-optimized coding regions can be designed by various differentmethods. This optimization may be performed using methods which areavailable on-line (e.g., GeneArt), published methods, or a company whichprovides codon optimizing services, e.g., DNA2.0 (Menlo Park, Calif.).One codon optimizing method is described, e.g., in US InternationalPatent Publication No. WO 2015/012924, which is incorporated byreference herein in its entirety. See also, e.g., US Patent PublicationNo. 2014/0032186 and US Patent Publication No. 2006/0136184. Suitably,the entire length of the open reading frame (ORF) for the product ismodified. However, in some embodiments, only a fragment of the ORF maybe altered. By using one of these methods, one can apply the frequenciesto any given polypeptide sequence, and produce a nucleic acid fragmentof a codon-optimized coding region which encodes the polypeptide.

A number of options are available for performing the actual changes tothe codons or for synthesizing the codon-optimized coding regionsdesigned as described herein. Such modifications or synthesis can beperformed using standard and routine molecular biological manipulationswell known to those of ordinary skill in the art. In one approach, aseries of complementary oligonucleotide pairs of 80-90 nucleotides eachin length and spanning the length of the desired sequence aresynthesized by standard methods. These oligonucleotide pairs aresynthesized such that upon annealing, they form double strandedfragments of 80-90 base pairs, containing cohesive ends, e.g., eacholigonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8,9, 10, or more bases beyond the region that is complementary to theother oligonucleotide in the pair. The single-stranded ends of each pairof oligonucleotides are designed to anneal with the single-stranded endof another pair of oligonucleotides. The oligonucleotide pairs areallowed to anneal, and approximately five to six of thesedouble-stranded fragments are then allowed to anneal together via thecohesive single stranded ends, and then they ligated together and clonedinto a standard bacterial cloning vector, for example, a TOPO® vectoravailable from Invitrogen Corporation, Carlsbad, Calif. The construct isthen sequenced by standard methods. Several of these constructsconsisting of 5 to 6 fragments of 80 to 90 base pair fragments ligatedtogether, i.e., fragments of about 500 base pairs, are prepared, suchthat the entire desired sequence is represented in a series of plasmidconstructs. The inserts of these plasmids are then cut with appropriaterestriction enzymes and ligated together to form the final construct.The final construct is then cloned into a standard bacterial cloningvector, and sequenced. Additional methods would be immediately apparentto the skilled artisan. In addition, gene synthesis is readily availablecommercially.

By “engineered” is meant that the nucleic acid sequences encoding theLebercilin protein described herein are assembled and placed into anysuitable genetic element, e.g., naked DNA, phage, transposon, cosmid,episome, etc., which transfers the Lebercilin sequences carried thereonto a host cell, e.g., for generating non-viral delivery systems (e.g.,RNA-based systems, naked DNA, or the like) or for generating viralvectors in a packaging host cell and/or for delivery to a host cells ina subject. In one embodiment, the genetic element is a plasmid. Themethods used to make such engineered constructs are known to those withskill in nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Green andSambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2012).

As used herein, the term “host cell” may refer to the packaging cellline in which a recombinant AAV is produced from a production plasmid.In the alternative, the term “host cell” may refer to any target cell inwhich expression of the coding sequence is desired. Thus, a “host cell,”refers to a prokaryotic or eukaryotic cell that contains exogenous orheterologous DNA that has been introduced into the cell by any means,e.g., electroporation, calcium phosphate precipitation, microinjection,transformation, viral infection, transfection, liposome delivery,membrane fusion techniques, high velocity DNA-coated pellets, viralinfection and protoplast fusion. In certain embodiments herein, the term“host cell” refers to the cells employed to generate and package theviral vector or recombinant virus. In other embodiments herein, the term“host cell” refers to cultures of ocular cells of various mammalianspecies for in vitro assessment of the compositions described herein.Still in other embodiments, the term “host cell” is intended toreference the ocular cells of the subject being treated in vivo for LCA.

As used herein, the term “ocular cells” refers to any cell in, orassociated with the function of, the eye. The term may refer to any oneof photoreceptor cells, including rod photoreceptors, conephotoreceptors and photosensitive ganglion cells, retinal pigmentepithelium (RPE) cells, Mueller cells, choroidal cells, bipolar cells,horizontal cells, and amacrine cells. In one embodiment, the ocularcells are the photoreceptor cells. In another embodiment, the ocularcells are cone photoreceptors. In another embodiment, the ocular cellsare rod photoreceptors.

In one embodiment, the nucleic acid sequence encoding Lebercilin furthercomprises a nucleic acid encoding a tag polypeptide covalently linkedthereto. The tag polypeptide may be selected from known “epitope tags”including, without limitation, a myc tag polypeptide, aglutathione-S-transferase tag polypeptide, a green fluorescent proteintag polypeptide, a myc-pyruvate kinase tag polypeptide, a His6 tagpolypeptide, an influenza virus hemagglutinin tag polypeptide, a flagtag polypeptide, and a maltose binding protein tag polypeptide.

In another aspect, an expression cassette comprising a nucleic acidsequence that encodes Lebercilin is provided. In one embodiment, thesequence is a codon optimized sequence. In another embodiment, the codonoptimized nucleic acid sequence is SEQ ID NO: 3 encoding humanLebercilin.

As used herein, an “expression cassette” refers to a nucleic acidmolecule which comprises the coding sequences for Lebercilin protein,promoter, and may include other regulatory sequences therefor, whichcassette may be packaged into the capsid of a viral vector (e.g., aviral particle). Typically, such an expression cassette for generating aviral vector contains the LCA5 sequences described herein flanked bypackaging signals of the viral genome and other expression controlsequences such as those described herein. For example, for an AAV viralvector, the packaging signals are the 5′ inverted terminal repeat (ITR)and the 3′ ITR. When packaged into the AAV capsid, the ITRs inconjunction with the expression cassette may be referred to herein asthe “recombinant AAV (rAAV) genome” or “vector genome”. In oneembodiment, an expression cassette comprises a codon optimized nucleicacid sequence that encodes Lebercilin protein. In one embodiment, thecassette provides the codon optimized LCA5 operatively associated withexpression control sequences that direct expression of the codonoptimized nucleic acid sequence that encodes Lebercilin in a host cell.In one embodiment, the vector genome is the sequence of nt 1-4379 of SEQID NO: 8. In another embodiment, the vector genome is the sequence of nt1-4368 of SEQ ID NO: 9. In yet another embodiment, the LCA5 codingsequence in either of the identified vector genomes is swapped withanother LCA5 coding sequence as described herein.

In another embodiment, an expression cassette for use in an AAV vectoris provided. In that embodiment, the AAV expression cassette includes atleast one AAV inverted terminal repeat (ITR) sequence. In anotherembodiment, the expression cassette comprises 5′ ITR sequences and 3′ITR sequences. In one embodiment, the 5′ and 3′ ITRs flank the codonoptimized nucleic acid sequence that encodes Lebercilin, optionally withadditional sequences which direct expression of the codon optimizednucleic acid sequence that encodes Lebercilin in a host cell. Thus, asdescribed herein, a AAV expression cassette is meant to describe anexpression cassette as described above flanked on its 5′ end by a 5′AAVinverted terminal repeat sequence (ITR) and on its 3′ end by a 3′ AAVITR. Thus, this rAAV genome contains the minimal sequences required topackage the expression cassette into an AAV viral particle, i.e., theAAV 5′ and 3′ ITRs. The AAV ITRs may be obtained from the ITR sequencesof any AAV, such as described herein. These ITRs may be of the same AAVorigin as the capsid employed in the resulting recombinant AAV, or of adifferent AAV origin (to produce an AAV pseudotype). In one embodiment,the ITR sequences from AAV2, or the deleted version thereof (AITR), areused for convenience and to accelerate regulatory approval. However,ITRs from other AAV sources may be selected. Where the source of theITRs is from AAV2 and the AAV capsid is from another AAV source, theresulting vector may be termed pseudotyped. Typically, the AAV vectorgenome comprises an AAV 5′ ITR, the Lebercilin coding sequences and anyregulatory sequences, and an AAV 3′ ITR. However, other configurationsof these elements may be suitable. A shortened version of the 5′ ITR,termed AITR, has been described in which the D-sequence and terminalresolution site (trs) are deleted. In other embodiments, the full-lengthAAV 5′ and 3′ ITRs are used. Each rAAV genome can be then introducedinto a production plasmid.

As used herein, the term “regulatory sequences”, “transcriptionalcontrol sequence” or “expression control sequence” refers to DNAsequences, such as initiator sequences, enhancer sequences, and promotersequences, which induce, repress, or otherwise control the transcriptionof protein encoding nucleic acid sequences to which they are operablylinked.

As used herein, the term “operably linked” or “operatively associated”refers to both expression control sequences that are contiguous with thenucleic acid sequence encoding the Lebercilin and/or expression controlsequences that act in trans or at a distance to control thetranscription and expression thereof.

In one aspect, a vector comprising any of the expression cassettesdescribed herein is provided. As described herein, such vectors can beplasmids of variety of origins and are useful in certain embodiments forthe generation of recombinant replication defective viruses as describedfurther herein.

A “vector” as used herein is a nucleic acid molecule into which anexogenous or heterologous or engineered nucleic acid transgene may beinserted which can then be introduced into an appropriate host cell.Vectors preferably have one or more origin of replication, and one ormore site into which the recombinant DNA can be inserted. Vectors oftenhave means by which cells with vectors can be selected from thosewithout, e.g., they encode drug resistance genes. Common vectors includeplasmids, viral genomes, and (primarily in yeast and bacteria)“artificial chromosomes.” Certain plasmids are described herein.

In one embodiment, the vector is a non-viral plasmid that comprises anexpression cassette described thereof, e.g., “naked DNA”, “naked plasmidDNA”, RNA, and mRNA; coupled with various compositions and nanoparticles, including, e.g., micelles, liposomes, cationic lipid—nucleicacid compositions, poly-glycan compositions and other polymers, lipidand/or cholesterol-based—nucleic acid conjugates, and other constructssuch as are described herein. See, e.g., X. Su et al, Mol.Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: Mar. 21, 2011;WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which areincorporated herein by reference. Such non-viral Lebercilin vector maybe administered by the routes described herein. The viral vectors, ornon-viral vectors, can be formulated with a physiologically acceptablecarrier for use in gene transfer and gene therapy applications.

In another embodiment, the vector is a viral vector that comprises anexpression cassette described therein. “Virus vectors” are defined asreplication defective viruses containing the exogenous or heterologousLCA5 nucleic acid transgene. In one embodiment, an expression cassetteas described herein may be engineered onto a plasmid which is used fordrug delivery or for production of a viral vector. Suitable viralvectors are preferably replication defective and selected from amongstthose which target ocular cells. Viral vectors may include any virussuitable for gene therapy, including but not limited to adenovirus;herpes virus; lentivirus; retrovirus; parvovirus, etc. However, for easeof understanding, the adeno-associated virus is referenced herein as anexemplary virus vector.

A “replication-defective virus” or “viral vector” refers to a syntheticor recombinant viral particle in which an expression cassette containinga gene of interest is packaged in a viral capsid or envelope, where anyviral genomic sequences also packaged within the viral capsid orenvelope are replication-deficient; i.e., they cannot generate progenyvirions but retain the ability to infect target cells. In oneembodiment, the genome of the viral vector does not include genesencoding the enzymes required to replicate (the genome can be engineeredto be “gutless”-containing only the transgene of interest flanked by thesignals required for amplification and packaging of the artificialgenome), but these genes may be supplied during production. Therefore,it is deemed safe for use in gene therapy since replication andinfection by progeny virions cannot occur except in the presence of theviral enzyme required for replication.

In another embodiment, a recombinant adeno-associated virus (rAAV)vector is provided. The rAAV compromises an AAV capsid, and a vectorgenome packaged therein.

The vector genome comprises, in one embodiment: (a) an AAV 5′ invertedterminal repeat (ITR) sequence; (b) a promoter; (c) a coding sequenceencoding a human Lebercilin; and (d) an AAV 3′ ITR. In anotherembodiment, the vector genome is the expression cassette describedherein. In one embodiment, the LCA5 sequence encodes a full lengthLebercilin protein. In one embodiment, the Lebercilin sequence is theprotein sequence of SEQ ID NO: 1. In another embodiment, the codingsequence is SEQ ID NO: 3 or a variant thereof.

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall nonenveloped, icosahedral virus with single-stranded linear DNAgenomes of 4.7 kilobases (kb) to 6 kb. Among known AAV serotypes areAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and others. TheITRs or other AAV components may be readily isolated or engineered usingtechniques available to those of skill in the art from an AAV. Such AAVmay be isolated, engineered, or obtained from academic, commercial, orpublic sources (e.g., the American Type Culture Collection, Manassas,Va.). Alternatively, the AAV sequences may be engineered throughsynthetic or other suitable means by reference to published sequencessuch as are available in the literature or in databases such as, e.g.,GenBank, PubMed, or the like. AAV viruses may be engineered byconventional molecular biology techniques, making it possible tooptimize these particles for cell specific delivery of nucleic acidsequences, for minimizing immunogenicity, for tuning stability andparticle lifetime, for efficient degradation, for accurate delivery tothe nucleus, etc.

Fragments of AAV may be readily utilized in a variety of vector systemsand host cells. Among desirable AAV fragments are the cap proteins,including the vp1, vp2, vp3 and hypervariable regions, the rep proteins,including rep 78, rep 68, rep 52, and rep 40, and the sequences encodingthese proteins. Such fragments may be used alone, in combination withother AAV serotype sequences or fragments, or in combination withelements from other AAV or non-AAV viral sequences. As used herein,artificial AAV serotypes include, without limitation, AAV with anon-naturally occurring capsid protein. Such an artificial capsid may begenerated by any suitable technique, using a novel AAV sequence of theinvention (e.g., a fragment of a vp1 capsid protein) in combination withheterologous sequences which may be obtained from another AAV serotype(known or novel), non-contiguous portions of the same AAV serotype, froma non-AAV viral source, or from a non-viral source. An artificial AAVserotype may be, without limitation, a chimeric AAV capsid, arecombinant AAV capsid, or a “humanized” AAV capsid. In one embodiment,a vector contains the AAV8 cap and/or rep sequences of the invention.See e.g., US patent application publication No. US2009/02270030,incorporated by reference herein.

The term “AAV” or “AAV serotype” as used herein refers to the dozens ofnaturally occurring and available adeno-associated viruses, as well asartificial AAVs. Among the AAVs isolated or engineered from human ornon-human primates (NHP) and well characterized, human AAV2 is the firstAAV that was developed as a gene transfer vector; it has been widelyused for efficient gene transfer experiments in different target tissuesand animal models. Unless otherwise specified, the AAV capsid, ITRs, andother selected AAV components described herein, may be readily selectedfrom among any AAV, including, without limitation, AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8 bp, AAV7M8 and AAVAnc80,variants of any of the known or mentioned AAVs or AAVs yet to bediscovered or variants or mixtures thereof. See, e.g., WO 2005/033321,which is incorporated herein by reference. In another embodiment, theAAV capsid is an AAV8 bp capsid, which preferentially targets bipolarcells. See, WO 2014/024282, which is incorporated herein by reference.In another embodiment, the AAV capsid is an AAV7m8 capsid, which hasshown preferential delivery to the outer retina. The AAV7m8 capsidnucleic acid sequence is reproduced in SEQ ID NO: 11 and amino acidsequence at SEQ ID NO: 12. See, Dalkara et al, In Vivo-DirectedEvolution of a New Adeno-Associated Virus for Therapeutic Outer RetinalGene Delivery from the Vitreous, Sci Transl Med 5, 189ra76 (2013), whichis incorporated herein by reference.

As used herein, an “AAV7m8 capsid” is a self-assembled AAV capsidcomposed of multiple AAV7m8 vp (variable protein) proteins. The AAV7m8vp proteins are typically expressed as alternative splice variantsencoded by a nucleic acid sequence of SEQ ID NO: 11 or a sequence atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 99% thereto, which encodes the vp1amino acid sequence of SEQ ID NO: 12. These splice variants result inproteins of different length of SEQ ID NO: 12. In certain embodiments,“AAV7m8 capsid” includes an AAV having an amino acid sequence which is99% identical to SEQ ID NO: 12.

In another embodiment, the rAAV capsid is selected from an AAV8 capsidor variant thereof, an AAV6 capsid or variant thereof, an AAV9 capsid orvariant thereof, an AAV7 capsid or variant thereof, an AAV5 capsid orvariant thereof, an AAV2 capsid or variant thereof, an AAV1 capsid orvariant thereof, an AAV3 capsid or variant thereof, and an AAV4 capsidor variant thereof. In one embodiment, a recombinant adeno-associatedvirus (rAAV) vector is provided which comprises an AAV7m8 capsid and anexpression cassette described herein, wherein said expression cassettecomprises nucleic acid sequences encoding Lebercilin, inverted terminalrepeat sequences and expression control sequences that direct expressionof Lebercilin in a host cell.

In still a further embodiment, a recombinant adeno-associated virus(AAV) vector is provided for delivery of the LCA5 constructs andoptimized sequences described herein. An adeno-associated virus (AAV)viral vector is an AAV DNase-resistant particle having an AAV proteincapsid into which is packaged nucleic acid sequences for delivery totarget cells. An AAV capsid is composed of 60 capsid (cap) proteinsubunits, VP1, VP2, and VP3, that are arranged in an icosahedralsymmetry in a ratio of approximately 1:1:10 to 1:1:20, depending uponthe selected AAV. AAVs may be selected as sources for capsids of AAVviral vectors as identified above. See, e.g., US Published PatentApplication No. 2007-0036760-A1; US Published Patent Application No.2009-0197338-A1; EP 1310571. See also, WO 2003/042397 (AAV7 and othersimian AAV), U.S. Pat. Nos. 7,790,449 and 7,282,199 (AAV8), WO2005/033321 and U.S. Pat. No. 7,906,111 (AAV9), and WO 2006/110689, andWO 2003/042397 (rh.10) and (Dalkara D, Byrne L C, Klimczak R R, Visel M,Yin L, Merigan W H, et al. In vivo-directed evolution of a newadeno-associated virus for therapeutic outer retinal gene delivery fromthe vitreous. Sci Transl Med (2013) 5(189):189ra76. doi:10.1126/scitranslmed.3005708) (AAV7m8). Each of these documents isincorporated herein by reference. These documents also describe otherAAV capsids which may be selected for generating AAV and areincorporated by reference. In some embodiments, an AAV cap for use inthe viral vector can be generated by mutagenesis (i.e., by insertions,deletions, or substitutions) of one of the aforementioned AAV capsids orits encoding nucleic acid. In some embodiments, the AAV capsid ischimeric, comprising domains from two or three or four or more of theaforementioned AAV capsid proteins. In some embodiments, the AAV capsidis a mosaic of Vp1, Vp2, and Vp3 monomers from two or three differentAAVs or recombinant AAVs. In some embodiments, an rAAV compositioncomprises more than one of the aforementioned Caps.

As used herein, relating to AAV, the term variant means any AAV sequencewhich is derived from a known AAV sequence, including those sharing atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 99% or greater sequence identity overthe amino acid or nucleic acid sequence. In another embodiment, the AAVcapsid includes variants which may include up to about 10% variationfrom any described or known AAV capsid sequence. That is, the AAV capsidshares about 90% identity to about 99.9% identity, about 95% to about99% identity or about 97% to about 98% identity to an AAV capsidprovided herein and/or known in the art. In one embodiment, the AAVcapsid shares at least 95% identity with an AAV capsid. When determiningthe percent identity of an AAV capsid, the comparison may be made overany of the variable proteins (e.g., vp1, vp2, or vp3). In oneembodiment, the AAV capsid shares at least 95% identity with the AAV7m8over the vp1, vp2 or vp3. In another embodiment, the capsid is an AAV8capsid with Y447F, Y733F and T494V mutations (also called“AAV8(C&G+T494V)” and “rep2-cap8(Y447F+733F+T494V)”), as described byKay et al, Targeting Photoreceptors via Intravitreal Delivery UsingNovel, Capsid-Mutated AAV Vectors, PLoS One. 2013; 8(4): e62097.Published online 2013 Apr. 26, which is incorporated herein byreference.

In one embodiment, it is desirable to utilize an AAV capsid, which showstropism for the desired target cell, e.g., photoreceptors (e.g., rodsand/or cones), RPE or other ocular cells. In one embodiment, the AAVcapsid is a tyrosine capsid-mutant in which certain surface exposedtyrosine residues are substituted with phenylalanine (F). Such AAVvariants are described, e.g., in Mowat et al, Tyrosine capsid-mutant AAVvectors for gene delivery to the canine retina from a subretinal orintravitreal approach, Gene Therapy 21, 96-105 (January 2014), which isincorporated herein by reference.

As used herein, “artificial AAV” means, without limitation, an AAV witha non-naturally occurring capsid protein. Such an artificial capsid maybe generated by any suitable technique, using a selected AAV sequence(e.g., a fragment of a vp1 capsid protein) in combination withheterologous sequences which may be obtained from a different selectedAAV, non-contiguous portions of the same AAV, from a non-AAV viralsource, or from a non-viral source. An artificial AAV may be, withoutlimitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAVcapsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein thecapsid of one AAV is replaced with a heterologous capsid protein, areuseful in the invention. In one embodiment, AAV2/5 and AAV2/8 areexemplary pseudotyped vectors.

In another embodiment, a self-complementary AAV is used.“Self-complementary AAV” refers a plasmid or vector having an expressioncassette in which a coding region carried by a recombinant AAV nucleicacid sequence has been designed to form an intra-moleculardouble-stranded DNA template. Upon infection, rather than waiting forcell mediated synthesis of the second strand, the two complementaryhalves of scAAV will associate to form one double stranded DNA (dsDNA)unit that is ready for immediate replication and transcription. See,e.g., D M McCarty et al, “Self-complementary recombinantadeno-associated virus (scAAV) vectors promote efficient transductionindependently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8,Number 16, Pages 1248-1254. Self-complementary AAVs are described in,e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of whichis incorporated herein by reference in its entirety.

The term “exogenous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein does not naturally occurin the position in which it exists in a chromosome, or host cell. Anexogenous nucleic acid sequence also refers to a sequence derived fromand inserted into the same host cell or subject, but which is present ina non-natural state, e.g. a different copy number, or under the controlof different regulatory elements.

The term “heterologous” as used to describe a nucleic acid sequence orprotein means that the nucleic acid or protein was derived from adifferent organism or a different species of the same organism than thehost cell or subject in which it is expressed. The term “heterologous”when used with reference to a protein or a nucleic acid in a plasmid,expression cassette, or vector, indicates that the protein or thenucleic acid is present with another sequence or subsequence which withwhich the protein or nucleic acid in question is not found in the samerelationship to each other in nature.

In still another embodiment, the expression cassette, including any ofthose described herein is employed to generate a recombinant AAV genome.

In one embodiment, the expression cassette described herein isengineered into a suitable genetic element (vector) useful forgenerating viral vectors and/or for delivery to a host cell, e.g., nakedDNA, phage, transposon, cosmid, episome, etc., which transfers the LCA5sequences carried thereon. The selected vector may be delivered by anysuitable method, including transfection, electroporation, liposomedelivery, membrane fusion techniques, high velocity DNA-coated pellets,viral infection and protoplast fusion. The methods used to make suchconstructs are known to those with skill in nucleic acid manipulationand include genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Sambrook et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

For packaging an expression cassette or rAAV genome or productionplasmid into virions, the ITRs are the only AAV components required incis in the same construct as the expression cassette. In one embodiment,the coding sequences for the replication (rep) and/or capsid (cap) areremoved from the AAV genome and supplied in trans or by a packaging cellline in order to generate the AAV vector.

Methods for generating and isolating AAV viral vectors suitable fordelivery to a subject are known in the art. See, e.g., U.S. Pat. Nos.7,790,449; 7,282,199; WO 2003/042397; WO 2005/033321, WO 2006/110689;and U.S. Pat. No. 7,588,772 B2]. In a one system, a producer cell lineis transiently transfected with a construct that encodes the transgeneflanked by ITRs and a construct(s) that encodes rep and cap. In a secondsystem, a packaging cell line that stably supplies rep and cap istransiently transfected with a construct encoding the transgene flankedby ITRs. In each of these systems, AAV virions are produced in responseto infection with helper adenovirus or herpesvirus, requiring theseparation of the rAAVs from contaminating virus. More recently, systemshave been developed that do not require infection with helper virus torecover the AAV—the required helper functions (i.e., adenovirus E1, E2a,VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesviruspolymerase) are also supplied, in trans, by the system. In these newersystems, the helper functions can be supplied by transient transfectionof the cells with constructs that encode the required helper functions,or the cells can be engineered to stably contain genes encoding thehelper functions, the expression of which can be controlled at thetranscriptional or posttranscriptional level.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, even ifsubsequently reintroduced into the natural system. Such polynucleotidescould be part of a vector and/or such polynucleotides or polypeptidescould be part of a composition, and still be isolated in that suchvector or composition is not part of its natural environment.

In yet another system, the expression cassette flanked by ITRs andrep/cap genes are introduced into insect cells by infection withbaculovirus-based vectors. For reviews on these production systems, seegenerally, e.g., Zhang et al., 2009, “Adenovirus-adeno-associated virushybrid for large-scale recombinant adeno-associated virus production,”Human Gene Therapy 20:922-929, the contents of which is incorporatedherein by reference in its entirety. Methods of making and using theseand other AAV production systems are also described in the followingU.S. patents, the contents of each of which is incorporated herein byreference in its entirety: U.S. Pat. Nos. 5,139,941; 5,741,683;6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753;7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. Seegenerally, e.g., Grieger & Samulski, 2005, “Adeno-associated virus as agene therapy vector: Vector development, production and clinicalapplications,” Adv. Biochem. Engin/Biotechnol. 99: 119-145; Buning etal., 2008, “Recent developments in adeno-associated virus vectortechnology,” J. Gene Med. 10:717-733; and the references cited below,each of which is incorporated herein by reference in its entirety.

The methods used to construct any embodiment of this invention are knownto those with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012). Similarly,methods of generating rAAV virions are well known and the selection of asuitable method is not a limitation on the present invention. See, e.g.,K. Fisher et al, (1993) J. Virol., 70:520-532 and U.S. Pat. No.5,478,745.

“Plasmids” generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. Many plasmids and other cloning and expression vectors that can beused in accordance with the present invention are well known and readilyavailable to those of skill in the art. Moreover, those of skill readilymay construct any number of other plasmids suitable for use in theinvention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentto those of skill from the present disclosure.

In one embodiment, the production plasmid is that described herein, oras described in WO2012/158757, which is incorporated herein byreference. Various plasmids are known in the art for use in producingrAAV vectors, and are useful herein. The production plasmids arecultured in the host cells which express the AAV cap and/or repproteins. In the host cells, each rAAV genome is rescued and packagedinto the capsid protein or envelope protein to form an infectious viralparticle.

In one aspect, a production plasmid comprising an expression cassettedescribed above is provided. In one embodiment, the production plasmidis that shown in SEQ ID NO: 8, and FIG. 1E-1F, which is termed p643.This plasmid is used in the examples for generation of the rAAV-humancodon optimized Lebercilin vector. Such a plasmid is one that contains a5′ AAV ITR sequence; a selected promoter; a polyA sequence; and a 3′ITR; additionally, it also contains a stuffer sequence, such as lambda.In a further embodiment, the stuffer sequence keeps the rAAV vectorgenome with a size between about 3 kilobases (kb) to about 6 kb, about4.7 kb to about 6 kb, about 3 kb to about 5.5 kb, or about 4.7 kb to 5.5kb. In one embodiment, a non-coding lambda stuffer region is included inthe vector backbone. An example of p643 which includes the Lebercilinencoding sequence can be found in SEQ ID NO: 8. In another embodiment,the production plasmid is that shown in FIG. 11A-11B and SEQ ID NO: 9.In another embodiment, the production plasmid is modified to optimizedvector plasmid production efficiency. Such modifications includeaddition of other neutral sequences, or deletion of portion(s) of or theentire lambda stuffer sequence to modulate the level of supercoil of thevector plasmid. Such modifications are contemplated herein. In otherembodiments, terminator and other sequences are included in the plasmid.

In certain embodiments, the rAAV expression cassette, the vector (suchas rAAV vector), the virus (such as rAAV), the production plasmidcomprises AAV inverted terminal repeat sequences, a codon optimizednucleic acid sequence that encodes Lebercilin, and expression controlsequences that direct expression of the encoded proteins in a host cell.In other embodiments, the rAAV expression cassette, the virus, thevector (such as rAAV vector), the production plasmid further compriseone or more of an intron, a Kozak sequence, a polyA,post-transcriptional regulatory elements and others. In one embodiment,the post-transcriptional regulatory element is Woodchuck Hepatitis Virus(WHP) Posttranscriptional Regulatory Element (WPRE).

The expression cassettes, vectors and plasmids include other componentsthat can be optimized for a specific species using techniques known inthe art including, e.g, codon optimization, as described herein. Thecomponents of the cassettes, vectors, plasmids and viruses or othercompositions described herein include a promoter sequence as part of theexpression control sequences. In another embodiment, the promoter iscell-specific. The term “cell-specific” means that the particularpromoter selected for the recombinant vector can direct expression ofthe optimized Lebercilin coding sequence in a particular ocular celltype. In one embodiment, the promoter is specific for expression of thetransgene in photoreceptor cells. In another embodiment, the promoter isspecific for expression in the rods and cones. In another embodiment,the promoter is specific for expression in the rods. In anotherembodiment, the promoter is specific for expression in the cones. In oneembodiment, the photoreceptor-specific promoter is a human rhodopsinkinase promoter. The rhodopsin kinase promoter has been shown to beactive in both rods and cones. See, e.g., Sun et al, Gene Therapy with aPromoter Targeting Both Rods and Cones Rescues Retinal DegenerationCaused by AIPL1 Mutations, Gene Ther. 2010 January; 17(1): 117-131,which is incorporated herein by reference in its entirety. In oneembodiment, the promoter is modified to add one or more restrictionsites to facilitate cloning.

In another embodiment, the promoter is a human rhodopsin promoter. Inone embodiment, the promoter is modified to include restriction on theends for cloning. See, e.g, Nathans and Hogness, Isolation andnucleotide sequence of the gene encoding human rhodopsin, PNAS,81:4851-5 (August 1984), which is incorporated herein by reference inits entirety. In another embodiment, the promoter is a portion orfragment of the human rhodopsin promoter. In another embodiment, thepromoter is a variant of the human rhodopsin promoter.

Other exemplary promoters include the human G-protein-coupled receptorprotein kinase 1 (GRK1) promoter (Genbank Accession number AY327580). Inanother embodiment, the promoter is a 292 nt fragment (positions1793-2087) of the GRK1 promoter (See, Beltran et al, Gene Therapy 201017:1162-74, which is hereby incorporated by reference in its entirety).In another preferred embodiment, the promoter is the humaninterphotoreceptor retinoid-binding protein proximal (IRBP) promoter. Inone embodiment, the promoter is a 235 nt fragment of the hIRBP promoter.In one embodiment, the promoter is the RPGR proximal promoter (Shu etal, IOVS, May 2102, which is incorporated by reference in its entirety).Other promoters useful in the invention include, without limitation, therod opsin promoter, the red-green opsin promoter, the blue opsinpromoter, the cGMP-β-phosphodiesterase promoter (Qgueta et al, IOVS,Invest Ophthalmol Vis Sci. 2000 December; 41(13):4059-63), the mouseopsin promoter (Beltran et al 2010 cited above), the rhodopsin promoter(Mussolino et al, Gene Ther, July 2011, 18(7):637-45); the alpha-subunitof cone transducin (Morrissey et al, BMC Dev, Biol, January 2011, 11:3);beta phosphodiesterase (PDE) promoter; the retinitis pigmentosa (RP1)promoter (Nicord et al, J. Gene Med, December 2007, 9(12):1015-23); theNXNL2/NXNL1 promoter (Lambard et al, PLoS One, Oct. 2010, 5(10):e13025),the RPE65 promoter; the retinal degeneration slow/peripherin 2(Rds/perph2) promoter (Cai et al, Exp Eye Res. 2010 August;91(2):186-94); and the VMD2 promoter (Kachi et al, Human Gene Therapy,2009 (20:31-9)). Each of these documents is incorporated by referenceherein in its entirety. In another embodiment, the promoter is selectedfrom human human EF1α promoter, rhodopsin promoter, rhodopsin kinase,interphotoreceptor binding protein (IRBP), cone opsin promoters(red-green, blue), cone opsin upstream sequences containing thered-green cone locus control region, cone transducing, and transcriptionfactor promoters (neural retina leucine zipper (Nr1) andphotoreceptor-specific nuclear receptor Nr2e3, bZIP).

In another embodiment, the promoter is a ubiquitous or constitutivepromoter. An example of a suitable promoter is a hybrid chicken β-actin(CBA) promoter with cytomegalovirus (CMV) enhancer elements, such as thesequence shown in FIG. 1E-1F. In another embodiment, the promoter is theCB7 promoter. Other suitable promoters include the human β-actinpromoter, the human elongation factor-1α promoter, the cytomegalovirus(CMV) promoter, the simian virus 40 promoter, and the herpes simplexvirus thymidine kinase promoter. See, e.g., Damdindorj et al, (August2014) A Comparative Analysis of Constitutive Promoters Located inAdeno-Associated Viral Vectors. PLoS ONE 9(8): e106472. Still othersuitable promoters include viral promoters, constitutive promoters,regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943].Alternatively a promoter responsive to physiologic cues may be utilizedin the expression cassette, rAAV genomes, vectors, plasmids and virusesdescribed herein. In one embodiment, the promoter is of a small size,under 1000 bp, due to the size limitations of the AAV vector. In anotherembodiment, the promoter is under 400 bp. Other promoters may beselected by one of skill in the art.

In a further embodiment, the promoter is selected from SV40 promoter,the dihydrofolate reductase promoter, and the phosphoglycerol kinase(PGK) promoter, rhodopsin kinase promoter, the rod opsin promoter, thered-green opsin promoter, the blue opsin promoter, the interphotoreceptor binding protein (IRBP) promoter and thecGMP-β-phosphodiesterase promoter, a phage lambda (PL) promoter, aherpes simplex viral (HSV) promoter, a tetracycline-controlledtrans-activator-responsive promoter (tet) system, a long terminal repeat(LTR) promoter, such as a RSV LTR, MoMLV LTR, BIV LTR or an HIV LTR, aU3 region promoter of Moloney murine sarcoma virus, a Granzyme Apromoter, a regulatory sequence(s) of the metallothionein gene, a CD34promoter, a CD8 promoter, a thymidine kinase (TK) promoter, a B19parvovirus promoter, a PGK promoter, a glucocorticoid promoter, a heatshock protein (HSP) promoter, such as HSP65 and HSP70 promoters, animmunoglobulin promoter, an MMTV promoter, a Rous sarcoma virus (RSV)promoter, a lac promoter, a CaMV 35S promoter, a nopaline synthetasepromoter, an MND promoter, or an MNC promoter. The promoter sequencesthereof are known to one of skill in the art or available publically,such as in the literature or in databases, e.g., GenBank, PubMed, or thelike.

In another embodiment, the promoter is an inducible promoter. Theinducible promoter may be selected from known promoters including therapamycin/rapalog promoter, the ecdysone promoter, theestrogen-responsive promoter, and the tetracycline-responsive promoter,or heterodimeric repressor switch. See, Sochor et al, An AutogenouslyRegulated Expression System for Gene Therapeutic Ocular Applications.Scientific Reports, 2015 Nov. 24; 5:17105 and Daber R, Lewis M., A novelmolecular switch. J Mol Biol. 2009 Aug. 28;391(4):661-70, Epub 2009 Jun.21 which are both incorporated herein by reference in their entirety.

In a further embodiment, the promoter is a chicken beta-actin promoterwith a nucleic acid sequence from nt 546 to nt 283 of SEQ ID NO. 8.

In other embodiments, the expression cassette, vector, plasmid and virusdescribed herein contain other appropriate transcription initiation,termination, enhancer sequences, efficient RNA processing signals suchas splicing and polyadenylation (polyA) signals; TATA sequences;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); introns;sequences that enhance protein stability; and when desired, sequencesthat enhance secretion of the encoded product. The expression cassetteor vector may contain none, one or more of any of the elements describedherein.

Examples of suitable polyA sequences include, e.g., a synthetic polyA orfrom bovine growth hormone (bGH), human growth hormone (hGH), SV40,rabbit β-globin (RGB), or modified RGB (mRGB). In a further embodiment,the poly A has a nucleic acid sequence from nt 3993 to nt 4200 of SEQ IDNO:8.

Examples of suitable enhancers include, e.g., the CMV enhancer, the RSVenhancer, the alpha fetoprotein enhancer, the TTR minimalpromoter/enhancer, LSP (TH-binding globulinpromoter/alpha1-microglobulin/bikunin enhancer), an APB enhancer, ABPSenhancer, an alpha mic/bik enhancer, TTR enhancer, en34, ApoE amongstothers. In one embodiment, the enhancer has a nucleic acid sequence fromnt 241 to nt 544 of SEQ ID NO: 8.

In one embodiment, a Kozak sequence is included upstream of theLebercilin coding sequence to enhance translation from the correctinitiation codon. In another embodiment, CBA exon 1 and intron areincluded in the expression cassette. In one embodiment, the Lebercilincoding sequence is placed under the control of a hybrid chicken β actin(CBA) promoter. This promoter consists of the cytomegalovirus (CMV)immediate early enhancer, the proximal chicken β actin promoter, and CBAexon 1 flanked by intron 1 sequences.

In another embodiment, the intron is selected from CBA, human betaglobin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen,ovalbumin, p53, or a fragment thereof.

In one embodiment, the expression cassette, the vector, the plasmid andthe virus contain a 5′ ITR, chicken beta-actin (CBA) promoter, CMVenhancer, CBA exon 1 and intron, human codon optimized Lebercilinsequence, bGH poly A and 3′ ITR. In a further embodiment, the expressioncassette includes nt 1 to 4379 of SEQ ID NO: 8. In yet a furtherembodiment, the 5′ ITR has a nucleic acid sequence from nt 1 to nt 130of SEQ ID NO: 8 and the 3′ITR has a nucleic acid sequence from nt 4250to nt 4379 of SEQ ID NO: 8. In a further embodiment, the CBA exon1 andintron has a nucleic acid sequence from nt 824 to nt 1795 of SEQ IDNO:8. In a further embodiment, the production plasmid has a sequence ofSEQ ID NO: 8, also shown in FIGS. 1E-1F. In a further embodiment, theproduction plasmid has a sequence of SEQ ID NO: 9, also shown in FIGS.1A-11B.

In another aspect, a method for treating Leber Congenital Amaurosiscaused by a defect in the lebercilin gene and/or restoring visualfunction in a subject having LCA comprises delivering to a subject inneed thereof a vector (such as rAAV) which encodes Lebercilin, asdescribed herein. In one embodiment, a method of treating a subjecthaving LCA with a rAAV described herein is provided.

By “administering” as used in the methods means delivering thecomposition to the target selected cell which is characterized by LCA.In one embodiment, the method involves delivering the composition bysubretinal injection to the RPE, photoreceptor cells or other ocularcells. In another embodiment, intravitreal injection to the subject isemployed. In another embodiment, subretinal injection to the subject isemployed. In still another method, intravascular injections, such asinjection via the palpebral vein may be employed. Still other methods ofadministration may be selected by one of skill in the art given thisdisclosure.

By “administering” or “route of administration” is delivery ofcomposition described herein, with or without a pharmaceutical carrieror excipient, of the subject. Routes of administration may be combined,if desired. In some embodiments, the administration is repeatedperiodically. The pharmaceutical compositions described herein aredesigned for delivery to subjects in need thereof by any suitable routeor a combination of different routes. In some embodiments, directdelivery to the eye (optionally via ocular delivery, subretinalinjection, intra-retinal injection, intravitreal, topical), or deliveryvia systemic routes is employed, e.g., intravascular, intraarterial,intraocular, intravenous, intramuscular, subcutaneous, intradermal, andother parental routes of administration. The nucleic acid molecules, theexpression cassette and/or vectors described herein may be delivered ina single composition or multiple compositions. Optionally, two or moredifferent AAV may be delivered, or multiple viruses [see, e.g., WO202011/126808 and WO 2013/049493]. In another embodiment, multiple virusesmay contain different replication-defective viruses (e.g., AAV andadenovirus), alone or in combination with proteins.

Also provided herein are pharmaceutical compositions. The pharmaceuticalcompositions described herein are designed for delivery to subjects inneed thereof by any suitable route or a combination of different routes.These delivery means are designed to avoid direct systemic delivery ofthe suspension containing the AAV composition(s) described herein.Suitably, this may have the benefit of reducing dose as compared tosystemic administration, reducing toxicity and/or reducing undesirableimmune responses to the AAV and/or transgene product.

In yet other aspects, these nucleic acid sequences, vectors, expressioncassettes and rAAV viral vectors are useful in a pharmaceuticalcomposition, which also comprises a pharmaceutically acceptable carrier,excipient, buffer, diluent, surfactant, preservative and/or adjuvant,etc. Such pharmaceutical compositions are used to express the optimizedLebercilin in the host cells through delivery by such recombinantlyengineered AAVs or artificial AAVs.

To prepare these pharmaceutical compositions containing the nucleic acidsequences, vectors, expression cassettes and rAAV viral vectors, thesequences or vectors or viral vector is preferably assessed forcontamination by conventional methods and then formulated into apharmaceutical composition suitable for administration to the eye. Suchformulation involves the use of a pharmaceutically and/orphysiologically acceptable vehicle or carrier, particularly one suitablefor administration to the eye, such as buffered saline or other buffers,e.g., HEPES, to maintain pH at appropriate physiological levels, and,optionally, other medicinal agents, pharmaceutical agents, stabilizingagents, buffers, carriers, adjuvants, diluents, surfactant, or excipientetc. For injection, the carrier will typically be a liquid. Exemplaryphysiologically acceptable carriers include sterile, pyrogen-free waterand sterile, pyrogen-free, phosphate buffered saline. A variety of suchknown carriers are provided in U.S. Pat. No. 7,629,322, incorporatedherein by reference. In one embodiment, the carrier is an isotonicsodium chloride solution. In another embodiment, the carrier is balancedsalt solution. In one embodiment, the carrier includes tween. If thevirus is to be stored long-term, it may be frozen in the presence ofglycerol or Tween20.

In certain embodiments, for administration to a human patient, the rAAVis suitably suspended in an aqueous solution containing saline, asurfactant, and a physiologically compatible salt or mixture of salts.Suitably, the formulation is adjusted to a physiologically acceptablepH, e.g., in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, orpH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about 7.28 toabout 7.32, for intrathecal delivery, a pH within this range may bedesired; whereas for intravitreal or subretinal delivery, a pH of 6.8 toabout 7.2 may be desired. However, other pHs within the broadest rangesand these subranges may be selected for other route of delivery.

A suitable surfactant, or combination of surfactants, may be selectedfrom among nonionic surfactants that are nontoxic. In one embodiment, adifunctional block copolymer surfactant terminating in primary hydroxylgroups is selected, e.g., such as Pluronic® F68 [BASF], also known asPoloxamer 188, which has a neutral pH, has an average molecular weightof 8400. Other surfactants and other Poloxamers may be selected, i.e.,nonionic triblock copolymers composed of a central hydrophobic chain ofpolyoxypropylene (poly(propylene oxide)) flanked by two hydrophilicchains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15(Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride),polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acidesters), ethanol and polyethylene glycol. In one embodiment, theformulation contains a poloxamer. These copolymers are commonly namedwith the letter “P” (for poloxamer) followed by three digits: the firsttwo digits×100 give the approximate molecular mass of thepolyoxypropylene core, and the last digit×10 gives the percentagepolyoxyethylene content. In one embodiment Poloxamer 188 is selected.The surfactant may be present in an amount up to about 0.0005% to about0.001% of the suspension.

In one example, the formulation may contain, e.g., buffered salinesolution comprising one or more of sodium chloride, sodium bicarbonate,dextrose, magnesium sulfate (e.g., magnesium sulfate.7H2O), potassiumchloride, calcium chloride (e.g., calcium chloride.2H2O), dibasic sodiumphosphate, and mixtures thereof, in water. Suitably, for intrathecaldelivery, the osmolarity is within a range compatible with cerebrospinalfluid (e.g., about 275 to about 290); see, e.g.,emedicine.medscape.com/article/2093316-overview. Optionally, forintrathecal delivery, a commercially available diluent may be used as asuspending agent, or in combination with another suspending agent andother optional excipients. See, e.g., Elliotts B® solution [LukareMedical]. In other embodiments, the formulation may contain one or morepermeation enhancers. Examples of suitable permeation enhancers mayinclude, e.g., mannitol, sodium glycocholate, sodium taurocholate,sodium deoxycholate, sodium salicylate, sodium caprylate, sodiumcaprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA.

In another embodiment, the composition includes a carrier, diluent,excipient and/or adjuvant. Suitable carriers may be readily selected byone of skill in the art in view of the indication for which the transfervirus is directed. For example, one suitable carrier includes saline,which may be formulated with a variety of buffering solutions (e.g.,phosphate buffered saline). Other exemplary carriers include sterilesaline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar,pectin, peanut oil, sesame oil, and water. The buffer/carrier shouldinclude a component that prevents the rAAV, from sticking to theinfusion tubing but does not interfere with the rAAV binding activity invivo.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The compositions according to the present invention may comprise apharmaceutically acceptable carrier, such as defined above. Suitably,the compositions described herein comprise an effective amount of one ormore AAV suspended in a pharmaceutically suitable carrier and/or admixedwith suitable excipients designed for delivery to the subject viainjection, osmotic pump, intrathecal catheter, or for delivery byanother device or route. In one example, the composition is formulatedfor intravitreal delivery. In one example, the composition is formulatedfor subretinal delivery.

In one exemplary specific embodiment, the composition of the carrier orexcipient contains 180 mM NaCl, 10 mM NaPi, pH7.3 with 0.0001%-0.01%Pluronic F68 (PF68). The exact composition of the saline component ofthe buffer ranges from 160 mM to 180 mM NaCl. Optionally, a different pHbuffer (potentially HEPES, sodium bicarbonate, TRIS) is used in place ofthe buffer specifically described. Still alternatively, a buffercontaining 0.9% NaCl is useful.

In the case of AAV viral vectors, quantification of the genome copies(“GC”), vector genomes (“VG”), or virus particles may be used as themeasure of the dose contained in the formulation or suspension. Anymethod known in the art can be used to determine the genome copy (GC)number of the replication-defective virus compositions of the invention.One method for performing AAV GC number titration is as follows:Purified AAV vector samples are first treated with DNase to eliminateun-encapsidated AAV genome DNA or contaminating plasmid DNA from theproduction process. The DNase resistant particles are then subjected toheat treatment to release the genome from the capsid. The releasedgenomes are then quantitated by real-time PCR using primer/probe setstargeting specific region of the viral genome (usually poly A signal).In another method the effective dose of a recombinant adeno-associatedvirus carrying a nucleic acid sequence encoding the optimized Lebercilincoding sequence is measured as described in S. K. McLaughlin et al, 1988J. Virol., 62:1963, which is incorporated by reference in its entirety.

As used herein, the term “dosage” can refer to the total dosagedelivered to the subject in the course of treatment, or the amountdelivered in a single unit (or multiple unit or split dosage)administration. The pharmaceutical virus compositions can be formulatedin dosage units to contain an amount of replication-defective viruscarrying the codon optimized nucleic acid sequences encoding Lebercilinas described herein that is in the range of about 1.0×10⁹ GC to about1.0×10¹⁵ GC per dose including all integers or fractional amounts withinthe range. In one embodiment, the compositions are formulated to containat least 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, or9×10⁹ GC per dose including all integers or fractional amounts withinthe range. In another embodiment, the compositions are formulated tocontain at least 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰,8×10¹⁰, or 9×10¹⁰ GC per dose including all integers or fractionalamounts within the range. In another embodiment, the compositions areformulated to contain at least 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, or 9×10¹¹ GC per dose including all integers orfractional amounts within the range. In another embodiment, thecompositions are formulated to contain at least 1×10¹², 2×10¹², 3×10¹²,4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², or 9×10¹² GC per dose includingall integers or fractional amounts within the range. In anotherembodiment, the compositions are formulated to contain at least 1×10¹³,2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, or 9×10¹³ GC perdose including all integers or fractional amounts within the range. Inanother embodiment, the compositions are formulated to contain at least1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴, 7×10¹⁴, 8×10¹⁴, or9×10¹⁴ GC per dose including all integers or fractional amounts withinthe range. In another embodiment, the compositions are formulated tocontain at least 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵, 7×10¹⁵,8×10¹⁵, or 9×10¹⁵ GC per dose including all integers or fractionalamounts within the range. In one embodiment, for human application thedose can range from 1×10¹⁰ to about 1×10¹² GC per dose including allintegers or fractional amounts within the range. All dosages may bemeasured by any known method, including as measured by oqPCR or digitaldroplet PCR (ddPCR) as described in, e.g., M. Lock et al, Hum Gene TherMethods. 2014 April; 25(2):115-25. doi: 10.1089/hgtb.2013.131, which isincorporated herein by reference.

In one embodiment, an aqueous suspension suitable for administration toan LCA patient is provided. The suspension comprises an aqueoussuspending liquid and about 1×10¹⁰ GC or viral particles to about 1×10¹²GC or viral particles per eye of a recombinant adeno-associated virus(rAAV) described herein useful as a therapeutic for LCA.

It may also be desirable to administer multiple “booster” dosages of thepharmaceutical compositions of this invention. For example, dependingupon the duration of the transgene within the ocular target cell, onemay deliver booster dosages at 6 month intervals, or yearly followingthe first administration. The fact that AAV-neutralizing antibodies werenot generated by administration of the rAAV vector should allowadditional booster administrations.

Such booster dosages and the need therefor can be monitored by theattending physicians, using, for example, the retinal and visualfunction tests and the visual behavior tests described in the examplesbelow. Other similar tests may be used to determine the status of thetreated subject over time. Selection of the appropriate tests may bemade by the attending physician. Still alternatively, the method of thisinvention may also involve injection of a larger volume ofvirus-containing solution in a single or multiple infection to allowlevels of visual function close to those found in wildtype retinas.

In another embodiment, the amount of the vectors, the virus and thereplication-defective virus described herein carrying the codonoptimized nucleic acid sequences encoding Lebercilin are in the range ofabout 1.0×10⁷ VG per eye to about 1.0×10¹⁵ VG per eye including allintegers or fractional amounts within the range. In one embodiment, theamount thereof is at least 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, or 9×10⁷ VG per eye including all integers or fractionalamounts within the range. In one embodiment, the amount thereof is atleast 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, or 9×10⁸VG per eye including all integers or fractional amounts within therange. In one embodiment, the amount thereof is at least 1×10⁹, 2×10⁹,3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, or 9×10⁹ VG per eye includingall integers or fractional amounts within the range. In one embodiment,the amount thereof is at least 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, or 9×10¹⁰ VG per eye including all integers orfractional amounts within the range. In one embodiment, the amountthereof is at least 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹,7×10¹¹, 8×10¹¹, or 9×10¹¹ VG per eye including all integers orfractional amounts within the range. In one embodiment, the amountthereof is at least 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹², 6×10¹²,7×10¹², 8×10¹², or 9×10¹² VG per eye including all integers orfractional amounts within the range. In one embodiment, the amountthereof is at least 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³, 6×10¹³,7×10¹³, 8×10¹³, or 9×10¹³ VG per eye including all integers orfractional amounts within the range. In one embodiment, the amountthereof is at least 1×10¹⁴, 2×10¹⁴, 3×10¹⁴, 4×10¹⁴, 5×10¹⁴, 6×10¹⁴,7×10¹⁴, 8×10¹⁴, or 9×10¹⁴ VG per eye including all integers orfractional amounts within the range. In one embodiment, the amountthereof is at least 1×10¹⁵, 2×10¹⁵, 3×10¹⁵, 4×10¹⁵, 5×10¹⁵, 6×10¹⁵,7×10¹⁵, 8×10¹⁵, or 9×10¹⁵ GC per dose including all integers orfractional amounts within the range. In one embodiment, the methodscomprises dose ranging from 1×10⁹ to about 1×10¹³ VG per eye per doseincluding all integers or fractional amounts within the range. Inanother embodiment, the method comprises delivery of the vector in anaqueous suspension. In another embodiment, the method comprisesadministering the rAAV described herein in a dosage of from 1×10⁹ to1×10¹³ GC in a volume about or at least 150 microliters, therebyrestoring visual function in said subject. All dosages may be measuredby any known method, including as measured by oqPCR or digital dropletPCR (ddPCR) as described in, e.g., M. Lock et al, Hum Gene Ther Methods.2014 April; 25(2):115-25. doi: 10.1089/hgtb.2013.131, which isincorporated herein by reference.

These above doses may be administered in a variety of volumes ofcarrier, excipient or buffer formulation, ranging from about 25 to about1000 microliters, including all numbers within the range, depending onthe size of the area to be treated, the viral titer used, the route ofadministration, and the desired effect of the method. In one embodiment,the volume of carrier, excipient or buffer is at least about 25 μL. Inone embodiment, the volume is about 50 μL. In another embodiment, thevolume is about 75 μL. In another embodiment, the volume is about 100μL. In another embodiment, the volume is about 125 μL. In anotherembodiment, the volume is about 150 μL. In another embodiment, thevolume is about 175 μL. In yet another embodiment, the volume is about200 μL. In another embodiment, the volume is about 225 μL. In yetanother embodiment, the volume is about 250 μL. In yet anotherembodiment, the volume is about 275 μL. In yet another embodiment, thevolume is about 300 μL. In yet another embodiment, the volume is about325 μL. In another embodiment, the volume is about 350 μL. In anotherembodiment, the volume is about 375 μL. In another embodiment, thevolume is about 400 μL. In another embodiment, the volume is about 450μL. In another embodiment, the volume is about 500 μL. In anotherembodiment, the volume is about 550 μL. In another embodiment, thevolume is about 600 μL. In another embodiment, the volume is about 650μL. In another embodiment, the volume is about 700 μL. In anotherembodiment, the volume is about 800 μL. In another embodiment, thevolume is between about 150 and 800 μL. In another embodiment, thevolume is between about 700 and 1000 pt. In another embodiment, thevolume is between about 250 and 500 pt.

In one embodiment, the viral constructs may be delivered in doses offrom at least 1×10⁹ to about least 1×10¹¹ GCs in volumes of about 1 μLto about 3 μL for small animal subjects, such as mice. For largerveterinary subjects having eyes about the same size as human eyes, thelarger human dosages and volumes stated above are useful. See, e.g.,Diehl et al, J. Applied Toxicology, 21:15-23 (2001) for a discussion ofgood practices for administration of substances to various veterinaryanimals. This document is incorporated herein by reference.

It is desirable that the lowest effective concentration of virus orother delivery vehicle be utilized in order to reduce the risk ofundesirable effects, such as toxicity, retinal dysplasia and detachment.Still other dosages in these ranges may be selected by the attendingphysician, taking into account the physical state of the subject,preferably human, being treated, the age of the subject, the LCA and thedegree to which the disorder, if progressive, has developed.

Yet another aspect described herein is a method for treating, retardingor halting progression of LCA in a mammalian subject. In one embodiment,an rAAV carrying the Lebercilin native, modified or codon optimizedsequence, preferably suspended in a physiologically compatible carrier,diluent, excipient and/or adjuvant, may be administered to a desiredsubject including a human subject. This method comprises administeringto a subject in need thereof any of the nucleic acid sequences,expression cassettes, rAAV genomes, plasmids, vectors or rAAV vectors orcompositions containing them. In one embodiment, the composition isdelivered subretinally. In another embodiment, the composition isdelivered intravitreally. In still another embodiment, the compositionis delivered using a combination of administrative routes suitable fortreatment of LCA, and may also involve administration via the palpebralvein or other intravenous or conventional administration routes.

For use in these methods, the volume and viral titer of each dosage isdetermined individually, as further described herein, and may be thesame or different from other treatments performed in the same, orcontralateral, eye. The dosages, administrations and regimens may bedetermined by the attending physician given the teachings of thisspecification. In one embodiment, the composition is administered in asingle dosage selected from those above listed in an affected eye. Inanother embodiment, the composition is administered as a single dosageselected from those above listed in a both affected eyes, eithersimultaneously or sequentially. Sequential administration may imply atime gap of administration from one eye to another from intervals ofminutes, hours, days, weeks or months. In another embodiment, the methodinvolves administering the compositions to an eye two or more dosages(e.g., split dosages). In another embodiment, multiple injections aremade in different portions of the same eye. In another embodiment, asecond administration of an rAAV including the selected expressioncassette (e.g., LCA5 containing cassette) is performed at a later timepoint. Such time point may be weeks, months or years following the firstadministration. Such second administration is, in one embodiment,performed with an rAAV having a different capsid than the rAAV from thefirst administration. In another embodiment, the rAAV from the first andsecond administration have the same capsid.

In still other embodiments, the compositions described herein may bedelivered in a single composition or multiple compositions. Optionally,two or more different AAV may be delivered, or multiple viruses [see,e.g., WO 2011/126808 and WO 2013/049493]. In another embodiment,multiple viruses may contain different replication-defective viruses(e.g., AAV and adenovirus).

In certain embodiments of the invention, it is desirable to performnon-invasive retinal imaging and functional studies to identify areas ofthe rod and cone photoreceptors to be targeted for therapy as well as totest the efficacy of treatment. In these embodiments, clinicaldiagnostic tests are employed to determine the precise location(s) forone or more subretinal injection(s). These tests may includeelectroretinography (ERG), perimetry, topographical mapping of thelayers of the retina and measurement of the thickness of its layers bymeans of confocal scanning laser ophthalmoscopy (cSLO) and opticalcoherence tomography (OCT), topographical mapping of cone density viaadaptive optics (AO), functional eye exam, Multi-electrode array (MEA),Pupillary Light Responses, etc, depending upon the species of thesubject being treated, their physical status and health and the dosage.In view of the imaging and functional studies, in some embodiments ofthe invention one or more injections are performed in the same eye inorder to target different areas of the affected eye. The volume andviral titer of each injection is determined individually, as furtherdescribed herein, and may be the same or different from other injectionsperformed in the same, or contralateral, eye. In another embodiment, asingle, larger volume injection is made in order to treat the entireeye. In one embodiment, the volume and concentration of the rAAVcomposition is selected so that only the region of damaged ocular cellsis impacted. In another embodiment, the volume and/or concentration ofthe rAAV composition is a greater amount, in order reach larger portionsof the eye, including non-damaged photoreceptors.

In another embodiment, the method includes performing additionalstudies, e.g., functional and imaging studies to determine the efficacyof the treatment. For examination in animals, such tests include retinaland visual function assessment via electroretinograms (ERGs) looking atrod and cone photoreceptor function, optokinetic nystagmus,pupillometry, water maze testing, light-dark preference, opticalcoherence tomography (to measure thickness of various layers of theretina), histology (retinal thickness, rows of nuclei in the outernuclear layer, immunofluorescence to document transgene expression, conephotoreceptor counting, staining of retinal sections with peanutagglutinin—which identifies cone photoreceptor sheaths).

Specifically for human subjects, following administration of a dosage ofa compositions described in this specification, the subject is testedfor efficacy of treatment using electroretinograms (ERGs) to examine rodand cone photoreceptor function, pupillometry visual acuity, contrastsensitivity color vision testing, visual field testing (Humphrey visualfields/Goldmann visual fields), perimetry mobility test (obstaclecourse), and reading speed test. Other useful post-treatment efficacytest to which the subject is exposed following treatment with apharmaceutical composition described herein are functional magneticresonance imaging (fMRI), full-field light sensitivity testing, retinalstructure studies including optical coherence tomography, fundusphotography, fundus autofluorescence, adaptive optics laser scanningophthalmoscopy, mobility testing, test of reading speed and accuracy,microperimetry and/or ophthalmoscopy. These and other efficacy tests aredescribed in U.S. Pat. No. 8,147,823; in co-pending International patentapplication publication WO 2014/011210 or WO 2014/124282, incorporatedby reference.

In one embodiment of the methods described herein, a one-timeintra-ocular delivery of a composition as described herein, e.g., an AAVdelivery of an optimized LCA5 cassette, is useful in treating LCA in asubject. In another embodiment of the methods described herein, aone-time intra-ocular delivery of a composition as described herein,e.g., an AAV delivery of an optimized LCA5 cassette, is useful intreating LCA in a subject at risk.

Thus, in one embodiment, the composition is administered before diseaseonset. In another embodiment, the composition is administered prior tothe initiation of vision impairment or loss. In another embodiment, thecomposition is administered after initiation of vision impairment orloss. In yet another embodiment, the composition is administered whenless than 90% of the rod and/or cones or photoreceptors are functioningor remaining, as compared to a non-diseased eye. In one embodiment,neonatal treatment is defined as being administered a Lebercilin codingsequence, expression cassette or vector as described herein within 8hours, the first 12 hours, the first 24 hours, or the first 48 hours ofdelivery. In another embodiment, particularly for a primate (human ornon-human), neonatal delivery is within the period of about 12 hours toabout 1 week, 2 weeks, 3 weeks, or about 1 month, or after about 24hours to about 48 hours. In another embodiment, the composition isdelivered after onset of symptoms. In one embodiment, treatment of thepatient (e.g., a first injection) is initiated prior to the first yearof life. In another embodiment, treatment is initiated after the first 1year, or after the first 2 to 3 years of age, after 5 years of age,after 11 years of age, or at an older age. In one embodiment, treatmentis initiated from ages about 4 years of age to about 12 years of age. Inone embodiment, treatment is initiated on or after about 4 years of age.In one embodiment, treatment is initiated on or after about 5 years ofage. In one embodiment, treatment is initiated on or after about 6 yearsof age. In one embodiment, treatment is initiated on or after about 7years of age. In one embodiment, treatment is initiated on or afterabout 8 years of age. In one embodiment, treatment is initiated on orafter about 9 years of age. In one embodiment, treatment is initiated onor after about 10 years of age. In one embodiment, treatment isinitiated on or after about 11 years of age. In one embodiment,treatment is initiated on or after about 12 years of age. However,treatment can be initiated on or after about 15, about 20, about 25,about 30, about 35, or about 40 years of age. In one embodiment,treatment in utero is defined as administering the composition asdescribed herein in the fetus. See, e.g., David et al, Recombinantadeno-associated virus-mediated in utero gene transfer gives therapeutictransgene expression in the sheep, Hum Gene Ther. 2011 April;22(4):419-26. doi: 10.1089/hum.2010.007. Epub 2011 Feb. 2, which isincorporated herein by reference.

In another embodiment, the composition is readministered at a laterdate. Optionally, more than one readministration is permitted. Suchreadministration may be with the same type of vector, a different viralvector, or via non-viral delivery as described herein. In oneembodiment, the vector is readministered to the patient to a differentportion of the initially injected retina. In one embodiment, the vectoris readministered to the patient to the same portion of the initiallyinjected retina.

In yet another embodiment, any of the above described methods isperformed in combination with another, or secondary, therapy. Thesecondary therapy may be any now known, or as yet unknown, therapy whichhelps prevent, arrest or ameliorate these mutations or defects or any ofthe effects associated therewith. The secondary therapy can beadministered before, concurrent with, or after administration of thecompositions described above. In one embodiment, a secondary therapyinvolves non-specific approaches for maintaining the health of theretinal cells, such as administration of neurotrophic factors,anti-oxidants, anti-apoptotic agents. The non-specific approaches areachieved through injection of proteins, recombinant DNA, recombinantviral vectors, stem cells, fetal tissue, or genetically modified cells.The latter could include genetically modified cells that areencapsulated.

In one embodiment, a method of generating a recombinant rAAV comprisesobtaining a plasmid containing an AAV expression cassette as describedabove and culturing a packaging cell carrying the plasmid in thepresence of sufficient viral sequences to permit packaging of the AAVviral genome into an infectious AAV envelope or capsid. Specific methodsof rAAV vector generation are described above and may be employed ingenerating a rAAV vector that can deliver the codon optimized LCA5 inthe expression cassettes and genomes described above and in the examplesbelow.

In certain embodiments of this invention, a subject has Leber congenitalamaurosis (LCA), for which the components, compositions and methods ofthis invention are designed to treat. As used herein, the term “subject”as used herein means a mammalian animal, including a human, a veterinaryor farm animal, a domestic animal or pet, and animals normally used forclinical research. In one embodiment, the subject of these methods andcompositions is a human. Still other suitable subjects include, withoutlimitation, murine, rat, canine, feline, porcine, bovine, ovine,non-human primate and others. As used herein, the term “subject” is usedinterchangeably with “patient”.

As used herein, the term “treatment” or “treating” is definedencompassing administering to a subject one or more compounds orcompositions described herein for the purposes of amelioration of one ormore symptoms of LCA. “Treatment” can thus include one or more ofreducing onset or progression of LCA, preventing disease, reducing theseverity of the disease symptoms, or retarding their progression,including the progression of blindness, removing the disease symptoms,delaying onset of disease or monitoring progression of disease orefficacy of therapy in a given subject.

It is to be noted that the term “a” or “an” refers to one or more. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively. While various embodiments in the specificationare presented using “comprising” language, under other circumstances, arelated embodiment is also intended to be interpreted and describedusing “consisting of” or “consisting essentially of” language.

As used herein, “disease”, “disorder” and “condition” are usedinterchangeably, to indicate an abnormal state in a subject.

As used herein, the term “about” or “—” means a variability of 10% fromthe reference given, unless otherwise specified.

The term “regulation” or variations thereof as used herein refers to theability of a composition to inhibit one or more components of abiological pathway.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art and by reference to published texts, whichprovide one skilled in the art with a general guide to many of the termsused in the present application.

EXAMPLES

The following examples are illustrative only and are not intended tolimit the present invention.

Example 1: Recombinant rAAV and In Vitro Expression Studies

Retinal gene transfer using the most thoroughly studied recombinant AAVserotype, AAV2, has been carried out in >310 eyes in human subjects, andin 29 different clinical trials (clinicaltrials.gov). These trialstarget diverse diseases including autosomal defects (RPE65 deficiency,retinitis pigmentosa due to MERTK mutations, choroideremia,achromatopsia), a mitochondrial disease (Leber's hereditary opticneuropathy), a complication of age-related macular degeneration(choroidal neovascularization) and end-stage retinal degeneration (usingoptogenetic therapy). In the majority of these studies (18/29 or 18 ofthe 22 studies that employed AAV and subretinal injection), the goal wasto target RPE cells efficiently. The net result has been a large body ofsafety data with respect to intra-ocular delivery of AAV. Subretinalinjection is the same surgical approach that will be necessary to targetphotoreceptors in LCA5 patients.

Tremendous efforts have been made to develop a path to treat LCA5 andother diseases involving primary photoreceptor defects based on theinformation obtained. Unfortunately, AAV2 vectors do not targetphotoreceptors efficiently, and, as mentioned above, photoreceptorscomprise the primary cell type in LCA5 and most other inherited retinaldegenerations. For this reason, AAV7m8, a vector generated byevolutionary design was selected. This vector has been shown to targetphotoreceptors efficiently in diverse species (mice and non-humanprimates (NHPs)) and using different routes of administration.

The efficacy reported herein includes improvements in the ability oftreated animals to navigate using visual cues, the restoration of visualpathways to the brain as shown by pupillometry, a reduction inphotoreceptor apoptosis, and the preservation of functionalphotoreceptors with morphology and markers characteristic of this celltype, including presence of rhodopsin in the outer retina. The treatedLca5gt/gt photoreceptors show thick outer nuclear layers with preservedouter segments with stacked outer segment discs. This is in markedcontrast to the untreated Lca5gt/gt retinas, which were reduced to asingle row of non-contiguous photoreceptor nuclei by 3 months of age 19.The improvements were not permanent. However, they persisted for atleast 3 months at which point there are no remaining photoreceptors inthe untreated Lca5gt/gt mouse. Electroretinography and MEA results showthat with successful transduction of photoreceptors, LCA5 gene therapyis capable of at least partially restoring responses mediated by bothrod and cone photoreceptors. The responses of the cells have near normalkinetics, including responses reflecting the activity of a variety ofganglion cell types as well as a reversal of the dominating melanopsinresponses observed in the untreated Lca5gt/gt retinas. The results arecomplementary with the pupillometry and visual behavior findings andwill provide the framework for future studies aiming to furthercharacterize and optimize the treatment effects (including studies ofvisual behavior in low illuminance conditions). These data provide hopethat a similar gene augmentation approach in humans as that used in theLca5gt/gt mouse could result in improved vision. It may be possible tofurther optimize the intervention to obtain an even more durable rescueeffect. Alterations of components of the transgene cassette (promoter,etc.), and areas of retina treated may lead to additional benefit.Dosing studies should identify the optimal dose for therapeutic effect.The fact that LCA5 null patients may retain photoreceptors throughadulthood (whereas photoreceptors are lost early in life in mice)suggests that there may be a wider window of opportunity in LCA5 humanscompared to Lca5−/− mice. Photoreceptors have been documented in LCA5null humans in the foveal outer nuclear layer, for up to 3 decades. Thisis important since successful gene therapy requires that the affectedcells be present. We were able to demonstrate that the retainedphotoreceptors in an adult with LCA5 mutations showed a similar temporalpattern of light responsiveness (albeit reduced in amplitude) asphotoreceptors from a normal-sighted individual. These results indicatethat the residual photoreceptors in LCA5 patients are functional,despite the structural and physiological deficits. The primary cilia ofiPSC-RPE derived from LCA5 mutant patients were much less numerous thanthose from the control cells. The facts that the ciliary defect inphotoreceptors in the Lca5gt/gt mouse can be corrected by geneaugmentation therapy and that the numbers of cilia can be increased tonormal levels, suggest that it may be possible to ameliorate the ciliarydefect present in humans with this condition.

A. Recombinant AAV

Recombinant AAVs were generated in the Center for Advanced Retinal andOcular Therapeutics (CAROT) using AAV7m8 capsid, which is known toinfect photoreceptors more efficiently than AAV2 (Dalkara D, Byrne L C,Klimczak R R, Visel M, Yin L, Merigan W H, et al. In vivo-directedevolution of a new adeno-associated virus for therapeutic outer retinalgene delivery from the vitreous. Sci Transl Med (2013) 5(189):189ra76.doi: 10.1126/scitranslmed.3005708.) The human (h) wildtypelebercilin-encoding optimized cDNA (hopt.LCA5) was custom-designed foroptimal codon usage and synthesized by DNA2.0 (Menlo Park, Calif.). TheLCA5 cDNA was placed under the control of a hybrid chicken β-actin (CBA)exon 1 flanked by intron 1 sequences and with cytomegalovirus (CMV)immediate early enhancer (FIGS. 1A and 1E). A bovine growth hormonepoly(A) followed the cDNA. A long stuffer sequence was included toprevent reverse packaging (i.e. of non-transgene containing) vector fromthe AAV2 inverted terminal repeats. The vectors were made by tripletransfection and formulated in excipient consisting ofphosphate-buffered saline (PBS) containing and 0.001% Pluronic F68(PF68). See, e.g. Mizukami, Hiroaki, et al. A Protocol for AAV vectorproduction and purification. Diss. Di-vision of Genetic Therapeutics,Center for Molecular Medicine, 1998. Control vectors incorporated theenhanced green fluorescent protein (eGFP) cDNA in place of the LCA5cDNA.

The rAAVs were tested for expression of the appropriate sized transgenicprotein by Western blot. 8431 cells were plated at 2×10⁶ cells per well.Two days after plating, cells were transduced with AAV7m8.hopt.LCA5 (orAAV7m8.CBA.EGFP as control) at 1×10⁵ or 5×10⁵ vg. 48 hours later, cellswere harvested and processed for electrophoresis and Western blot.Antibodies include an LCA5 rabbit polyclonal antibody (Proteintech,Rosemont, Ill.) and the signals are quantified, with each value iscorrected for background and protein loading differences throughnormalization with the GAPDH immunosignal.

The AAV7m8.CBA.hopt.LCA5 virus is able to drive efficient expression ofthe LCA5 transgene in 8431 cells. Production of the predicted ˜81 kDALebercilin protein after infection with AAV7m8.CBA.hopt.LCA5demonstrates a dose-dependent responses.

Example 2: LCA Mouse Model Lca5−/− Mouse Studies

Development of a proof-of-concept of gene augmentation therapy in theLca5gt/gt mouse model entails several challenges: 1) because retinaldegenerative changes begin very early and progress rapidly, interventionmust be carried out in neonatal mice; 2) since this is aphotoreceptor-specific disease, recombinant AAV vectors must be employedthat target photoreceptors efficiently. The AAV2 vector used extensivelyin animal and human studies to target RPE cells does not targetphotoreceptors as efficiently as other AAV serotypes as shown bytransduction comparisons of different serotypes after infection withequivalent doses. Ideally, therapeutics should be developed that couldultimately progress to human clinical trials; and 3) outcome measuresmust be developed that accurately identify and quantify improvements inretinal and visual function, which is so low at baseline that it isdifficult to score. Here we used a recombinant AAV vector (AAV7m8)designed by directed evolution, to deliver a codon optimized humanlebercilin-encoding cDNA. By using AAV7m8 to deliver LCA5 to thediseased photoreceptors early in life, we show that gene augmentationtherapy results in both structural improvement of the Lca5gt/gt mouseretina and functional improvement of its vision.

Lebercilin localizes to connecting cilia of photoreceptor cells (22).The connecting cilium is a transition zone between the photoreceptorcell body inner segment and the antennae-like outer segments thatsupports selective transport of proteins and membrane vesicles. Theconnecting cilium is thus the conduit supporting bi-directional proteintrafficking along ciliary microtubule tracks, or intraflagellartransport (IFT). Using both quantitative affinity proteomics (affinitypurification, mass spectrometry and bioinformatics analyses) and agenetically engineered mouse model, Boldt et al demonstrated that LCA5mutations interfere with IFT, (22) thereby causing an early-onset defectin photoreceptor outer segment development and a failure to correctlytraffic two different proteins expressed specifically in photoreceptors,arrestin and opsin. Knockout of the Lca5 gene in mice resulted in aretinal degeneration phenotype. The Lca5−/− mice develop patches ofde-pigmented retina, never develop outer segments and lack cone and rodERG responses to light. There is an early and rapidly progressiveretinal degeneration and only a single (sickly) row of nuclei is presentin the ONL by 2 months of age. (22) Thus, the Lca5−/− mice were utilizedas an animal model for LCA.

Adult Lca5^(gt/gt) (Lca5−/−) mice were purchased from Jackson Labs (BarHarbor, Me.) and a line was generated by brother-sister crossings.Verification of the genotype of all animals used in the study wasperformed (see Supplementary Methods). Mice were on a 12-hourlight/12-hour dark cycle, and food/water was provided ad libitum. Thestudies were performed in compliance with federal and institutionalregulations.

Subretinal injections were carried out unilaterally in neonatal mice asdescribed previously (28) in cohorts of pups. Anesthesia in mice atpostnatal day 5 (PN5) was hypothermia. At postnatal day 15 (PN15),animals were anesthetized with ketamine/xylazine. Table 1 shows thenumber of animals used per cohort.

Intravitreal injections of AAV7m8 were also carried out since thisvector had been shown previously to penetrate the mouse retina from thevitreal aspect to target photoreceptors. (25) AAV7m8.CBA.hopt.LCA5 wasinjected at a total of 9.20×10⁹ vg in 1 μl in cohort 1 (Table 1). Theinjection solution contained 5% v/v of AAV7m8.CBA.EGFP so that the areaof injection could be identified with certainty at later time pointsthrough presence of enhanced green fluorescent protein (EGFP).Additional animals received sham injection (cohort 2), receivedinjection of AAV7m8.CBA.EGFP alone (cohort 3), or were maintaineduninjected as controls (cohort 4). After injection, pups were returnedto their mothers until the time of weaning.

TABLE 1 Cohorts of neonatal Lca5−/− mice injected at the designatedpostnatal day (PN) and studied in vivo. Group Eye #1 Eye #2 AnimalNumber Testing material ROA Testing material ROA Number PN5 1 excipientintravitreal NA sham 5 + 4 2 excipient subretinal NA sham 7 + 4 3AAV7m8.hopt.LCA5 intravitreal NA sham 10 + 8  (+5% AAV7m8.GFP) 4AAV7m8.hopt.LCA5 + subretinal NA sham 17 + 8  5% AAV7m8.GFP) PN15 5excipient intravitreal NA sham 13 6 excipient subretinal NA sham 6 7AAV7m8.hopt.LCA5 intravitreal NA sham 13 (+5% AAV7m8.GFP) 8AAV7m8.hopt.LCA5 subretinal NA sham 11 (+5% AAV7m8.GFP) ROA—route ofadministration.

Ophthalmoscopy was carried out about 1 month post injection to verifythat media were clear, retinas were not detached, and thus that therehad been no surgical complications. Any animals that had corneal orvitreal opacities were excluded from further study.

Cohorts of mice were bred and studied, and injections were carried outat early postnatal (PN5) and juvenile (PN15) time points (Table 1).Injections were found largely without complications. After injections atPN5, the majority of the animals were found to be free of corneal orvitreal opacities that would interfere with further testing. The fewanimals with opacities were excluded from further analyses.

The AAV7m8.CβA.hopt-LCA5 virus was able to drive efficient expression ofthe human LCA5 transgene in mouse retina (FIGS. 1A and 1B) followingboth intravitreal (IVT) and subretinal (SR) administration (FIGS. 1C and1D, respectively). Western blot analysis shows production of thepredicted ˜81 kDa LCA5 protein after intra-ocular delivery withAAV7m8.CβA.hopt-LCA5 in Lca5gt/gt and wild type mice at PN20 (FIG. 1B).Immunofluorescence analysis shows presence of lebercilin protein in theONL, inner segments (IS) and connecting cilia (CC) in wild type adultretina (FIG. 1E). After injecting AAV7m8.CβA.hopt-LCA5 intravitreally orsubretinally in Lca5gt/gt retinas, lebercilin is found in CC as well asIS and ONL (FIGS. 1G and 1H). In contrast, in sham-injected Lca5gt/gtretina. there is no lebercilin (FIG. 1F). In addition, after IVTinjection, a substantial number of Muller cells produce lebercilin (FIG.1G).

Example 3: Tests of Retinal/Visual Function

Tests of visual and retinal function included a light-cued water maze(2-3 months post injection), pupillometry (2.5 months post injection),multi-electrode array (3 months post injection) and electroretinographyutilizing the mice indicated or described in Example 2. Retinas werethen evaluated for histopathology and immunofluorescence as described inExample 4. A thorough tissue analysis was also followed.

A. Electroretinography

We recorded ERGs from 8 Lca5gt/gt mice, 5 wild-type animals, and 16Lca5gt/gt mice whose left eyes had been injected with excipient and theright eyes with AAV7m8. CβA.hopt-LCA5 on PN5 intravitreally. None of theeyes of Lca5gt/gt uninjected controls or Lca5gt/gt eyes injected withexcipient (a total of 32 eyes) produced detectable ERG responses. Out of16 eyes injected with AAV7m8. CβA.hopt-LCA5, four showed ERG responsesto dim flashes of light in the dark-adapted state consistent withrod-mediation of the signals, as well as mixed rod-cone responses tobrighter flashes of light that resembled a smaller, scaled version ofwild-type (WT) mixed rod-cone ERG waveforms (Figure S2). Light-adaptedERGs were also detectable in these eyes suggesting cone-mediation of thephotopic responses. Detectable ERGs post-treatment ranged in amplitudefrom 20 to 25% the WT amplitudes (Figure S2). The results suggestrestoration of both rod and cone photoreceptor function after genetherapy in some of the animals. The inconsistency of the full-field ERGfindings likely reflect incomplete retinal coverage and/or tissue damage(cataracts, unresolved detachment, etc.) sustained after thesetechnically challenging subretinal injections, performed in the earlypost-natal period. The results, however variable in magnitude orinfrequent, were dramatically different from the undetectable ERGsobserved in untreated Lca5gt/gt eyes. B. Water maze navigation study

Water maze testing was performed to evaluate each animal's ability toidentify the chamber containing a submerged platform in a 5-chamberwater maze (FIG. 11 ). The apparatus was maintained in a room withoutextraneous light and the light source was placed directly over theplatform prior to the test. The dark-adapted mouse was released in thecenter of the maze and given 60 seconds, without interruption, to findand place all four paws on the lit platform.

Training was carried out in room light (about 200 Lux) prior to darkadapting the mice and carrying out the test under dim light procedures.During the training, if the mouse was unable to find the platform at theend of the 60 seconds, the experimenter guided the mouse to theplatform. For each trial, the light and platform were placed in adifferent chamber using random selection.

Training pass criteria were defined as the ability of the mouse toindependently enter the correct chamber, without deviating into adifferent chamber, and mount the platform within 60 seconds in more than5 of 9 sequential trials. All mice were trained for 5 days regardless ofthe day that training pass criteria were met. Testing was carried outfor 4 days using the same procedure as that used in training but with aseries of filters to further dim the light source. Through use offilters the luminances were: 1.06×10⁵, 8.69×10³, 5.87×10² scot cd m², orwith the light off, the luminance was 0 scot cd m².

Animals were trained and then tested with the light-cued water maze at2-2.5 months of age. Light-cued water maze test results showed that theAAV7m8.CBA.hopt.LCA5 subretinally or intravitreally injected groupsperformed significantly better than controls. (Table 2B; see values inBOLD; FIG. 3 ). Table 2A shows the raw data, which are numbers ofanimals analyzed in each cohort and then tested under the designatedlight levels using the water maze. Table 2B shows the results ofstatistical analyses using one-way analysis of variance (ANOVA). Thetreated animals were able to pass the test at 8.69×10³ scot cd m²whereas animals from the control excipient-injected cohort were not(p<0.01).

TABLE 2 Results of water maze testing in Lca5−/− mice. Mean (SD) forPass % Light Level (scot cd m²) (A) Intervention n 1.06 × 10⁵ 8.69 × 10³5.87 × 10² 0 Excipient (PBS) 5 53.3 31.1 31.1 13.3 Intravitreal at PN5(36.36) (9.29) (21.38) (9.29) Excipient (PBS) 13 46.15 33.31 17.08 33.32Intravitreal at PN15 (17.5) (17.57) (9.74) (13.64) Excipient (PBS) 773.0 34.9 22.2 14.3 Subretinal at PN5 (16.78) (16.28) (16.96) (12.35)AAV7m8-hopt-LCA5 (+5% GFP) 10 70.0 52.3 40.0 15.5 intravitreal at PN5(17.43) (13.93) (21.09) (13.03) AAV7m8-hopt-LCA5 (+5% GFP) 13 53.8528.19 18.78 27.75 intravitreal at PN15 (24.80) (17.35) (11.45) (11.64)AAV7m8-hopt-LCA5 (+5% GFP) 17 88.9 77.1 47.7 14.4 subretinal at PN5(16.2) (15.9) (23.8) (8.6) (B) P-value for Pairwise comparison LightLevel (scot cd m²) with PBS control group^(†) 1.06 × 10⁵ 8.69 × 10³ 5.87× 10² 0 AAV7m8-hopt-LCA5 0.24 0.01 0.46 0.74 intravitreal at PN5 vs.Excipient intravitreal at PN5 AAV7m8-hopt-LCA5 0.37 0.46 0.69 0.48intravitreal at PN15 vs. Excipient intravitreal at PN15 AAV7m8-hopt-LCA50.042 0.00001 0.018 0.981 subretinal at PN5 vs. Excipient subretinal atPN5

C. Pupillary Light Reflex

The amplitudes of pupillary constriction were measured during a seriesof 10 light flashes per eye. Flash intensity was 1,000 scot lux. PLRswere defined as having a maximum amplitude of pupil constrictionresponse within the 0.6-1.2 second interval following the flash that wasmore than 3 standard deviations of the pre-stimulus diameter. Ascientist masked to the treatment paradigm evaluated the data from eachanimal prior to analyses to be sure that each pupil was trackedaccurately. If not, that particular animal was excluded from furtheranalyses.

For statistical analyses, normalized pupil diameters from the treated(right) and contralateral sham-injected control (left) Lca5−/− eyes werecompared at each light intensity. By dividing the normalized amplitudeof constriction of the treated eye with that of the untreated eye, thepercentage of the Maximum Differential Amplitude of Response (MDAR)could be obtained. Higher percentage of the MDAR indicated a strongerresponse to intervention. Additional age-matched untreated Lca5−/− andwildtype (C57Bl/6) mice were used as positive and negative controls,respectively. The MDARs of untreated Lca5−/− and C57Bl/6 mice were about0 since there was minimal difference between the two eyes.

Additional analyses evaluated the magnitudes of amplitude of pupillaryconstriction in the right eyes of the different cohorts of mice. Thus,the amplitudes of constriction could be compared between treated anduntreated Lca5−/− mice and untreated C57Bl/6 mice.

Analyses of amplitudes of the pupillary light reflex generated bystimulation of the treated vs control eyes revealed a significant(p<0.05) improvement in amplitude of the pupillary reflex in eyes ofLca5−/− mice that were treated either subretinally or intravitreallywith AAV7m8.hopt-LCA5 compared to untreated control Lca5−/− animals(FIG. 2 ). There was significantly improved Pupillary Light Reflex (PLR)was detected in animals injected at PN5 by either route of delivery(FIGS. 2A, 2B, 2H, and 2I) and at PN15 with subretinal delivery,although there was also a trend to improvement with intravitrealdelivery (FIGS. 2H and 2I).

D. Multi-electrode Array

Multi-electrode array (MEA) testing was carried out on 5 animals 2.5-3.5months after they had received unilateral intravitreal injection withAAV7m8.CMV/CBA.hopt.LCA5 (about 9.87×10¹⁰ vg/eye) spiked with 5% (v/v)AAV7m8.CBA.GFP. Contralateral eyes were sham-injected and used asnegative controls. Five untreated age-matched wildtype (WT) C57Bl/6 miceserved as positive controls. Retinas from light-adapted mice weredissected under red light and mounted ganglion cell side down in theperforated MEA chamber. Presence of GFP in the explants confirmedexposure of photoreceptors to AAV. Calibrated full field flashes of 455nm light (efficiency of pigment excitation about 40%:rhodopsin andM-opsin; about 0.2%:S-opsin) of different intensities were used forlight stimulation (2 s flashes at 0.1 Hz or 50 ms flashes at 4 Hz). Datawere analyzed using custom code in Matlab (MatLab, Natick, Mass.); spikesorting was performed in Plexon Offline Sorter (Plexon, Dallas, Tex.).AAV7m8.CβA.hopt-LCA5 at PN5 were probed using multi-electrode array(MEA) analyses. The rationale for using MEA is that it measures theoutput signal of the retina sent to the brain and thus, in addition totesting photoreceptor function, also provides information about retinalwiring. Of the five retinas/animals tested with MEA, two had had clearlydetectable rod and cone ERGs (see a representative record in Figure S2),one had a residual small, ˜10 □V ERG, and two did not have anydetectable ERG. Three out of 5 AAV7m8.CBA.hopt.LCA5-injected retinas hadstrong light responses, one showed median responses and 1 showed minimalresponse in MEA testing (FIG. 5A-D). Responses became detectable atscotopic intensities (42-112 photons*s⁻¹*μm⁻², or on average, 8.28e1hv/μm²; 455 nm) and strong responses were observed through the brightestphotopic intensity of 2.00e9 photons*s⁻¹*μm⁻². Assuming collecting areafor rods and cones for end-on illumination to be around 1 μm2 at thewavelength of peak sensitivity 23, 24 (note that both in ERG and our MEAexperiments light enters the retina from the ganglion cell side) andefficiency of stimulation by 455 nm light of rhodopsin and of M- andS-cone opsins to be around 60%, 50% and 0.3% respectively 25-27 the dimlight generating clearly detectable responses in our experiments shouldproduce less than 70 photoisomerizations per second per cell in rods,less than 60 in M-cones, and around 0.3 photoisomerizations per secondin S-cones. Data from suction pipette recording show that at this rateof pigment excitation rods can generate detectable light responses(response amplitude above 20% of the maximum) while cone response isexpected to be at least 20 times smaller under the most favorableconditions (dark adapted Mcones in rod-transducing knockout retinas) andshould be undetectable in rod dominated mouse retina 25, 26, 28 (notethat collecting area for side-on illumination in suction pipetterecording is around 0.5 μm-2 for rods and 0.2 μm-2 for cones). Thusobservation of light responses at the dimmer intensity range in ourexperiments proves recovery of rod function in the treated Lca5gt/gtretinas. Responses at the brighter end of intensities should originatein cones (the brightest intensity should be more than capable of drivingboth M- and S-cones by producing around 1.00e9 and 6.00e6photoisomerizations per second per cell in M- and S-cones respectively).As expected for rod/cone driven responses, after exposure to thebrightest stimulation series at the end of the 1st intensity series run(light sensitive retinas were subjected to at least 2 intensity seriesruns ranging from the dim scotopic to the brightest photopic intensitiesin ˜0.5 log increments), scotopic responses disappeared, but photopicresponses were not significantly affected (FIGS. 5C and D). WT andtreated Lca5gt/gt retinas were also responsive to 4 Hz flickerstimulation at the intensities expected to drive cone responses (Datanot shown). Retinas from sham-injected contralateral eyes showed minimalto abolished responses with high spontaneous firing and prominentmelanopsin responses.

Slow melanopsin driven responses were also detected in bothAAV7m8.CβA.hopt-LCA5-treated Lca5gt/gt retinas and retinas from wildtype mice (manifesting as elevated firing rate at the start of the 2ndflash due to the slow recovery after the 1st flash in a series) ofmelanopsin response appears to be muted in treated retinas compared tothe untreated ones.

TABLE 3 Summary of MEA responses in treated and control Lca5−/− micenumber of treatment/outcome records comments treated Lca5−/− retina/ 4 3retina with responses nearly light responsive undistinguishable from WT,one with weaker but still prominent responses treated Lca5−/−retina/ 2one with weaker spontaneous firing not light responsive untreatedLca5−/− (left 6 all responding Lca5−/− retinas (right) retinacontrol)/not light have corresponding control from the responsive lefteye WT/light responsive 5 intensity range from 40 to 2e9 photons perum{circumflex over ( )}2 per second All retinas (except one treatedLca5−/− retina) demonstrated strong spontaneous firing, when tested formelanopsin-driven responses (long interflash interval to allow for slowrecovery). Untreated Lca5−/− retinas demonstrated strongmelanopsin-driven responses.

Two of the treated Lca5−/− retinas had signs of post-injection injuryand did not show light responses despite strong spontaneous firing.

Responses of the AAV7m8.CBA.hopt.LCA5-treated retinas were similar tothose of untreated wildtype retinas. One treated retina tested after theshortest post injection period demonstrated reduced light sensitivityand absence of OFF- and sustained ON-responses (red downward triangleson FIG. 5D). Development of the OFF-responses appears to require longertime post injection compared to the ON-responses, consistent withincreased variability in the OFF-response amplitudes observed for 10 thetreated retinas. Function of all ganglion cell types identifiable in theWT retinas under fullfield stimulation was detected in the Lca5gt/gtAAV-treated retinas after spike sorting (Figure S10). Strong responseswere observed under scotopic and photopic stimulation, up to thebrightest intensities (almost 100 mW/cm²). As expected for rod/conedriven responses, after exposure to the brightest light, scotopicresponses disappeared but photopic responses were not significantlyaffected (data not shown).

Responses in AAV7m8.hopt.LCA5-treated retinas became detectable atscotopic intensities (42-112 photons*s-1*μm-2, or on average, 8.28e1hv/μm2; 455 nm) and strong responses were observed through the brightestphotopic intensity of 2.00e9 photons*s-1*μm-2. Retinas fromsham-injected contralateral eyes showed minimal to abolished responses(Data not shown. Control Lca5−/−). Responses in AAV7m8.hopt.LCA5-treatedretinas were similar to those in retinas of wildtype mice. Flickerresponses showing that after exposure to the brightest light (2E9hv/cm2), photopic responses were not significantly affected (Data notshown, Lca5−/− treated). Flicker response intensity series (going from3.53E2-1.52E6 hv/cm2 of representative AAV7m8.CBAhopt.LCA5-treatedretina showed that as the light intensity increases, the amplitude ofresponse increases. Testing of rod and cone endurance responses afterthe brightest flashes showed persistence of the cone response, similarto that found in wildtype retinas. Flicker response testing showssimilar responses in treated Lca5−/− retinas compared to retinas ofwildtype mice. Firing rate of rods and cones in treated Lca5−/− retinasapproaches that of wildtype retinas (as opposed to the control untreatedLca5−/− retinas) as a function of light intensity.

Response kinetics were similar to those in WT retinas (Data not shown).Light sensitivity was slightly lower in treated Lca5−/− vs the mostsensitive WT retinas where first responses were detected at 8-21photons*s⁻¹*μm⁻² (Data not shown). All ganglion cell types identifiablein the WT retinas under full field stimulation (ON-, OFF andON/OFF-types) were detected in the Lca5−/− treated retinas after spikesorting. WT and treated Lca5−/− retinas were responsive to 4 Hz flickerstimulation at 3.53e2-2.00e9 photons*s⁻¹*μm⁻² (Data not shown). Alluntreated Lca5−/− retinas showed slow melanopsin driven responses atbrighter intensities which were absent in the light sensitive treatedLca5−/− retinas. A summary of the findings is shown in Table 3.

Though results obtained with electroretinography and MEA experimentsdemonstrated restoration of rod and cone function in at least someanimals, these methods were inadequate for assessment of interventionefficiency in a majority of animals since only a quarter of treated miceproduced useful ERG signals. Therefore we based assessment ofintervention efficacy on two additional approaches, analyses ofpupillary light reflexes (PLR) and visual behavior, which offered,first, higher sensitivity than electrophysiology, and, second, proofthat treatment of the Lca5gt/gt retina with AAV7m8. CβA.hopt-LCA5results in light-induced signal relayed beyond the retina, throughvisual pathways to the brain.

Pupillary light reflexes (PLR) are dependent upon relay of signalsinitially from photoreceptors to retinal ganglion cells, then to theEdinger-Westphal nucleus in the brain, and finally back to the pupillarysphincter muscle via the ciliary ganglion, which controls iris diameter.Analyses of the PLR following stimulation of the treated vs. controleyes revealed a significant (p<0.05) increase in amplitude of the PLR ineyes of Lca5gt/gt mice treated with AAV7m8.CβA.hopt-LCA5 compared tosham-injected controls (FIG. 2C). There was a significantly improved PLRdetected in animals injected at PN5 either intravitreally orsubretinally (FIGS. 2A-2B and 2E-2F) such that near-wild type responseswere observed (FIG. 2D). Statistically significant improvement of theamplitude of the PLR was also demonstrated through comparisons of themaximum differential amplitude of response (MDAR; FIG. 2G-2I; FIG. 51 ).Animals receiving AAV7m8.CβA.hopt-LCA5 by either IVT or SR delivery alsoshowed significantly improved PLR as quantified by MDAR (FIGS. 2H and2I).

Since PLRs demonstrate retinal signaling and function but do not provideinformation on formed vision, we used a light-cued water maze test tomeasure functional vision as described above. The majority of untreatedwildtype (normal-sighted) mice were able to navigate the water mazesuccessfully at all light levels whereas untreated Lca5gt/gt wereseverely impaired (Table 2, FIG. 3A). Results from water maze testingshowed that both IVT and subretinally AAV7m8.CβA.hopt-LCA5 injectedLca5gt/gt animals performed better than uninjected controls in astatistically significant manner under at least one light condition.(Table 2; FIG. 3 ). The animals treated subretinally at PN5 showed asignificantly improved pass rate at every lighting test condition(1.06E+05, 8.69E+03, and 5.87E+02 scot cd m-2), than controlexcipient-injected cohort (p<0.01). When animals were injected at PN15(IVT or SR), improvements were minimal (FIG. 3 ). To confirm that miceused only the given light cue, testing was performed under no lightcondition (0.00 scot cd m2) and over 90% of mice failed the test(including normal-sighted mice; FIG. 3B).

Example 4: Ocular Histology, Histopathology, Immunofluorescence andTUNEL Assay

Histologic rescue of Lca5gt/gt photoreceptors after treatment withAAV7m8.CβA.hopt-LCA5 at PN5 was assessed both through evaluation ofmolecules relevant to proper function of the phototransduction cascadeand though structural measures. Animals as described in Example 2 and 3were euthanized at 3 months of age. Eyes were enucleated and retinalsections evaluated by modifications of methods described by Boldt et al.(22) Tissue was fixed with 4% paraformaldehyde in PBS and thencryoprotected in 30% sucrose/PBS prior to freezing and generatingcryosections. Histopathology was analyzed by examination of4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific,Philadelphia, Pa.)-stained sections and/or by staining with hematoxylinand eosin. For immunofluorescence studies, sections were incubated withanti-lebercilin (1:300, (12, 22) in the presence of blocking solution,washed and then treated with Cy3-conjugatioed secondary antibodies.Additional antibodies used were anti-rhodopsin (1:500, LeicoTechnologies), anti-red/green cone opsin (1:250, Chemicon),anti-acetylated tubulin (1:1,000, Sigma-Aldrich). Stained sections werecover-slipped with Citifluor mounting medium containing DAPI (ElectronMicroscopy Services, Hatsfield, Pa.). TUNEL staining was carried outusing a terminal deoxynucleotidyl transferase (TdT) dUTP nick-endlabeling (TUNEL) assay kit following manufacturer's recommendations(Vector Laboratories, Burlingame, Calif.). Sections were evaluated witha Zeiss Axio Imager M2 microscope equipped with epifluorescence andAxio-Vision 4.6 software and with a confocal laser-scanning microscope(Olympus Fluoview 1000, Center Valley, Pa., USA). Transmission electronmicroscopy (TEM) was carried out on designated tissue samples using aFEI-Tecnai T12 S/TEM.

Immunofluorescence analysis of eyes injected with AAV7m8-hopt-LCA5 atPN5 and analyzed at PN15 showed Lebercilin co-localized with the base oftubulin-positive outer segments. After intravitreal injection,Lebercilin was distributed throughout the retina, which was nearlydevoid of photoreceptors at PN95. In contrast, Lebercilin was absent inuntreated PN15 and PN95 Lca5−/− retinas.

Hematoxylin and eosin-stained treated and control retinal sections atPN40 vs. PN99 comparing results after intravitreal (IVT) or subretinal(SR) injection of AAV7m8.hopt-LCA5 (with 5% v/v AAV7m8.eGFP) werecompared with sham injection. Micrographs of hematoxylin andeosin-stained (H&E) retina showed that inner/outer segments (IS/OS) andONL are preserved through the 3 months timepoint (PN99) after either IVTor SR injection of AAV7m8.hopt-LCA5 (Data not shown). In contrast,sham-injected control retinas lack such layers at PN99 (Data not shown).Further, lebercilin was detected in the treated, but not in sham treatedcontrol retinas (Data not shown). Persistent expression of rhodopsin inthe ONL was also confirmed by immunofluorescence analysis (Data notshown) but only seen in treated eyes.

The thickness of photoreceptor cell layers in retinas that were treatedby IVT or SR injection with AAV7m8.CβA.hopt-LCA5 at PN5 wassignificantly greater than in control retinas (FIG. 4 ). There was alsoa noticeable border in thickness between the areas of retinas exposed toor unexposed to AAV in retinas treated by subretinal injection (Data notshown). The preservation of photoreceptor layers persisted through thelatest timepoint (PN99). The increased thickness was due to an increasednumber of rows in the outer nuclear layer, and presence of inner andouter segments (FIG. 4 ). Consistent with this, there was a much higherproportion of dying photoreceptors in control compared toAAV.LCA5-treated retinas as judged by TUNEL labeling, particularlywithin the first month after intervention (Data not shown).

Evaluation of Lca5−/− mouse retinas injected on PN5 with AAV7m8.hLCA5 at1-3 months post injection reveals increased thickness of the outernuclear layer (FIG. 5A and FIG. 9 ). Immunofluorescence analysisrevealed that Lebercilin co-localized with the base of tubulin-positiveouter segments in AAV7m8.hOP.LCA5-treated Lca5−/− retinas (FIG. 1M).There were numerous TUNEL-positive cells in the first month of life inuntreated Lca5−/− retinas, but significantly fewer inAAV7m8.hOPT.LCA5-treated retinas (FIGS. 6B and 6C). The number ofTUNEL-positive cells decreased by the time the animals are 3 months ofage (FIGS. 6D and 6E). The majority of cells that degenerate in theuntreated Lca5−/− retina over time in control (untreated) retinas werephotoreceptors, as shown by the fact that rhodopsin-positive cells werefound only in the AAV-treated retinas (FIG. 6B) Immunofluorescenceanalysis confirmed this result (see DAPI-stained ONL), and showedincreased rhodopsin persisting through the 3 month time point (FIG. 6D).There was evidence of light-mediated changes in position ofphototransduction-specific molecules after injection withAAV7m8.hopt.LCA5 (FIG. 10 ).

Transmission electron microscopy (TEM) of AAV7m8.hopt.LCA5-treatedretinas showed elaboration of outer segments complete with stacked outersegment disks, 9+0 microtubule arrays typical of primary cilia (FIG. 7B)and connecting cilia (FIG. 7 ). In contrast, untreated Lca5−/− retinaslacked both connecting cilia and outer segments. The untreated retinaslack outer segments and show massive degeneration of photoreceptors withonly pyknotic nuclei and remnants of photoreceptor organelles. Onlydisorganized, degenerating photoreceptor cell bodies were present incontrol untreated or sham-injected retinas.

Light stimulation of dark-adapted adult Lca5gt/gt retinas treated at PN5with AAV7m8.hopt-LCA5 resulted into normal translocation patterns ofphototransduction proteins into outer segments. Arrestin translocatesproperly after light exposure in AAV7m8.hopt-LCA5-treated retinas. Suchactivity cannot be assessed in the control retinas due to thedegeneration of IS/OS. The data reflect restoration of theintraflagellar transport defect in the mouse retina after delivery ofthe wild type (WT) hLCA5 cDNA.

Development of proof-of-concept of gene augmentation therapy in theLca5−/− mouse model entails several challenges: 1) Because retinaldegenerative changes progress rapidly and early in life, interventionmust be carried out in neonatal mice; 2) Since this is aphotoreceptor-specific disease, recombinant AAV vectors must be employedthat target photoreceptors efficiently. The AAV2 vector that has beenused extensively in animal and human studies to target RPE cells, doesnot target photoreceptors as efficiently as other AAVs. (23, 24).Ideally, reagents should be used that could ultimately be used in humanclinical trials; and 3) Outcome measures must be developed that reflectimprovements in retinal and visual function, levels that are so low atbaseline that they are difficult to measure. Here a recombinant AAVdesigned by directed evolution, AAV7m8, (25) was used to test forefficacy after delivery of the native human Lebercilin-encoding cDNA.This vector has been shown by others to efficiently targetphotoreceptors after intra-vitreal delivery. (25-27). By using AAV7m8 todeliver the hLCA5 cDNA to the diseased photoreceptors early in life,gene augmentation therapy resulted in both structural and functionalimprovement of the Lca5−/− mouse retina and of vision.

In Examples 3 and 4, it was demonstrated that AAV7m8-mediated geneaugmentation therapy in the Lca5−/− mouse rescued retinal and visualfunction and also retinal structure.

The efficacy reported in the present example included the improvedability of this model to navigate using visual cues, restoration of rodand cone photoreceptor responses as shown by Multi-electrode array(MEA), a reduction in apoptotic cell death (and thus preservation ofphotoreceptors), and presence of cell biologic and physical featurescharacteristic of normally functioning photoreceptors, such as presenceof rhodopsin in the outer retina and development of normal-appearingouter segments, with stacked outer segment discs. MEA results showedthat given successful injection and enough post injection time toexpress the transgenic protein and restore function of rod/cone outersegments, gene therapy restored degenerated retinal cells to a statenearly indistinguishable from the WT conditions. Further, delivery ofwildtype LCA5 protected against degeneration of photoreceptors. Whileuntreated Lca5−/− retina was reduced to a single row of sicklyphotoreceptors by 3 months of age, the AAV-treated regions had a thickouter nuclear later complete with inner and outer segments. The gainspersisted at least 3 months—a very significant finding especiallyconsidering that by this age, there were no remaining photoreceptors inthe untreated Lca5−/− mouse retina. The data suggested that a similargene augmentation approach in humans as that used in the Lca5−/− mousecould result in improved vision.

Example 5: Human Studies: Pupillary Light Reflex Testing

Testing was carried out after obtaining written informed consent on anIRB approved protocol (#815348). Pupil responses were recordedsimultaneously in both eyes with a Procyon P3000 pupillometer andPupilFit6 software (Monmouthshire, UK). Pupillary responses to lightwere recorded after presentation of 10 lux green light stimuli to theright eye for 0.2 seconds followed by dark intervals. An infraredsensitive camera that captures video images at 25 frames per second,allowing the pupil diameter of both eyes to be measured every 40 ms.

Pupillary light reflex testing was carried out to determine whetherthere was any evidence of function in the residual photoreceptorspresent in an adult with homozygous LCA5 mutations. As shown in FIG. 8 ,pupillary light reflexes were present in this individual with the sametemporal sequence as those in a normal-sighted individual. However, theamplitudes of response were diminished considerably compared to thenormal-sighted individual.

Example 6: Induced Pluripotent Stem Cell (iPSC) Models of LCA5

To aid in translating the mouse rescue studies to human, we studiedhuman induced pluripotent stem cell (iPSC) lines that were generated forstudy from individuals with normal vision and those affected with LCAdue to LCA5 mutations. Recently, iPSC-derived retinal pigmentedepithelium (RPE) has been shown to recapitulate the functional phenotypeof polarized epithelium, with secretion, gene expression and maturationcharacteristics comparative to fetal and adult RPEs 29. These cells showgreat promise for understanding disease mechanisms associated withdefects in cilia biology 30, 31. Here, we differentiated iPSCs into RPEcells (FIG. 12A), a process which allowed evaluation of ciliated cellsin a shorter timespan and more efficiently than ifneuronal/photoreceptor cells had been generated (Figure S12). Weevaluated LCA5 gene expression and show that LCA5 mRNA is expressed inunaffected iPSC-derived RPEs and the level of expression of LCA5 in LCApatient RPEs is significantly reduced compared to controls (FIG. 12B).When ciliary distribution was measured, there were significantly fewercilia present on the iPSC-RPE derived from the LCA5 patient than on theRPEs from the normal-sighted individual (FIG. 12D-E). Treatment of theLCA5-iPSC-RPE with AAV7m8.hopt-LCA5 led to production of lebercilinprotein (FIG. 12C) and resulted in comparable cilia numbers in theAAV7m8.LCA5 treated and normal-sighted individual RPE cells (FIG.12D-F).

Peripheral blood monocytes (PBMCs) were collected after signed informedconsent (IRB approved protocol #808828) from two different probands withLCA5 mutations. One proband, JB605, was a compound heterozygote for LCA5Gln279Stop CAG>TAG het and Lys172de14ctcAAAG het; The other, JB590, washomozygous for LCA5 c.835C>T p.Q279X. The wildtype cells were fromindividual PBWT4.6. Induced pluripotent stem cell lines were generatedfrom each of the three individuals. PBMCs were cultured in expansionmedia consisting of QBSF-60 (Invitrogen, Carlsbad, Calif.) mediasupplemented with cytokines and hormone. The media was replenished every2-3 days for a period of 7-9 days until the cells entered a stage ofexponential growth.

For reprogramming, expanded PBMCs were transduced with the rTTAlentivirus and doxycycline inducible “stem cell cassette” in thelentivirus vector delivering OCT4, KLF4, SOX2, and cMyc cDNA andmicroRNA 302/367 cluster driven by the TetO/CMV promoter. Cells weregrown in expansion media supplemented with polybrene (5 μg/ml;Sigma-Aldrich, St. Louis, Mo.). The cells were incubated for 20-24 h at37° C. Infected cells were then washed, and placed in expansion mediasupplemented with 1 μg/ml of doxycycline (DOX). After 48 h, cells wereresuspended in Iscove's modified Dolbecco's medium (IMDM) with 10% fetalbovine serum (FBS), penicillin/streptomycin, L-glutamine,beta-mercaptoethanol, nonessential amino acids, 4 ng/ml of basicfibroblast growth factor (bFGF), and 1 μg/ml of DOX (Sigma-Aldrich, St.Louis, Mo.) then moved onto matrigel-coated (BD Biosciences, San Jose,Calif.) mouse embryonic fibroblast plates (MEF plates). Cells remainedin this media for 10 days, then were transferred to human embryonic stemcell (hESC) media (DMEM/F12, 20% knockout serum replacement,nonessential amino acid, 4 ng/ml bFGF, 0.001% beta-mercaptoethanol,penicillin/streptomycin, L-glutamine, and 1 μg/ml of DOX (Invitrogen,Carlsbad, Calif.). After 4 weeks, iPSC-like colonies were manuallypicked and expanded on matrigelcoated MEF plates for 6 passages, thentransitioned to 0.1% gelatin-coated MEF plates for a minimum of 15passages. Characterization of iPSCs was based on surface antigenexpression measured by flow cytometry using antibodies against SSEA3+,SSEA4+, TRA-1-60, and TRA-1-81 (Biolegend, San Diego, Calif.), andRT-qPCR analysis included pluripotency expression markers: DMNT3B,ABCG2, REX1, OCT4, SOX2, NANOG, cMYC, KLF4. Karyotyping of iPSCs wascarried out by G banding to produce a visible karyotype. The ability ofthe cells to differentiate into multiple different lineages was carriedout using a PCR germ layer assay (Qiagen. Germantown, Md., USA).

The characterized iPSCs were differentiated into RPE by activation ofthe Wnt signaling pathway, inhibition of the fibroblast growth factorsignaling pathway, and inhibition of the Rho-associated, coiled-coilcontaining protein kinase signaling pathway. Expanded pigmented cellswere purified by plate adhesion on 8-chamber slides on geltrex matrix.Enriched cells were cultured until they developed a cobblestoneappearance with cuboidal shape. The characteristics of iPS-RPE wereconfirmed by gene expression, immunocytochemistry, and microscopy. Cilialengths were measured after fixing and staining for ARL13B andPericentrin. Cilia lengths were measured in three dimensions usingcustom-designed software designed by the Wistar Imaging Facility (WistarInstitute, Philadelphia, Pa.)

Cilia are evaluated in the control and LCA5 iPSC-RPE cell lines fordensity and length. There are significantly fewer cilia present on theRPE cells derived from the two LCA5 patients than on the wildtype cellline. In addition, the cilia on the LCA5-derived lines are significantlyshorter than those in the wildtype cells.

It was previously observed that LCA5 patients can retain photoreceptorsincluding in the foveal outer nuclear layer, for up to 3 decades. (21,29). Since successful gene therapy requires that the cells be present(even if they are sickly), LCA5 photoreceptors may be amenable totreatment. The fact that the instant examples demonstrated that retainedphotoreceptors in an adult with LCA5 showed a similar temporal patternof light responsiveness (albeit reduced amplitude) as photoreceptorsfrom a normal-sighted individual is encouraging. These results furthersupported the fact that the residual photoreceptors in these abnormalretinas were viable despite their structural and functional deficits.Further, the cilia from the LCA patient-derived iPSC-RPE cells weresignificantly shorter than those from the wildtype control cells. Thefact that the ciliary defect in photoreceptors in the Lca5−/− mousecould be in the corrected by gene augmentation therapy, suggested thatit might ameliorate the ciliary defect present in humans with thiscondition. Studies aiming to correct the ciliary defect in vitro iniPSC-derived RPE cells are currently in process.

Gene augmentation therapy in humans with LCA5 is under investigation totest for safety and efficacy thereof. Besides the need to developappropriate outcome measures for this severe blinding condition, theinstant application provided solutions to several problems in planninghuman clinical trials, including developing constraints for achievingrescue? In the Lca5−/− mouse, the examples demonstrated rescue in bothneonatal (PN5) and juvenile (PN15) mice. An intervention was notobserved at later stages of the disease because of the earlyphotoreceptor loss in this mouse model. The abnormalities of developmentin the mouse model (coinciding with degeneration of photoreceptors)suggested that there were developmental components of the disease. Therewere likely developmental components in the human condition as well. Asmentioned above, in humans with LCA5, there is evidence of persistenceof some macular photoreceptors even in adults. (21) Investigation isundergoing to determine how to improve the function of thosephotoreceptors as well as to determine whether the visual pathways canbe resuscitated in humans with LCA5 based on the fact that there hasbeen success in resuscitating cortical vision in humans enrolled inRPE65 gene therapy clinical trials even after suffering for decades withlimited vision. (4, 30, 31)

Example 7: Genotyping of the Lca5−/− Mouse

Genomic DNA was PCR-amplified using the following oligos: LCA-13F6(common F) GCCTGTTCCTGCTTGCTTAC (enclosed as SEQ ID NO: 13); LCA-13R2(WT Reverse) TGCTTTCCAAAGTAAGCACAAA (enclosed as SEQ ID NO: 14)

en2.8r (mutant Reverse) CCTGGCCTCCAGACAAGTAG (enclosed as SEQ ID NO:15). PCR was run for 35 cycles with an annealing temperature of 50° C.and an extension step at 72° C. The predicted products are 353 bp(wildtype) and 439 bp (Lca5−/−).

Example 8: Intravitreal Injection of AAV7m8.hLCA5 Restores PhotoreceptorFunction in Lca5−/− Mice to Nearly WT Levels

Early onset vision loss results from mutations in LCA5, a gene whichencodes Lebercilin, a protein critical for the health and function ofphotoreceptors. Because there can be relative preservation ofphotoreceptors, LCA5 disease may be amenable to gene augmentationtherapy. The possibility that gene augmentation therapy usingintravitreal delivery of AAV7m8.CMV/CBA.hLCA5 restores photoreceptor andretinal function using multi electrode array (MEA) in an Lca5 mousemodel (Lca5−/−) was tested.

Postnatal day 5 Lca5−/− mice were injected intravitreally withAAV7m8.CMV/CBA.hLCA5 (approximately 9.87×10¹⁰ vg/eye) mixed with 5%(v/v) AAV7m8.CBA.GFP. Contralateral eyes were uninjected and used asnegative control. 3 months after the intervention and under lightadapted conditions, Lca5−/− retinas were dissected under red light andmounted ganglion cell side down in the perforated MEA chamber. The samedissection and preparation procedure was done for untreated age-matchedwild type mice (C57Bl/6) as a positive control. Presence of GFPconfirmed exposure of photoreceptors to the AAV. Calibrated full fieldflashes of 455 nm light in the scotopic and photopic intensity ranges(10 2 s flashes at 0.1 Hz or 400 50 ms flashes at 4 Hz) were used forlight stimulation (blue light was used to maximize chances of responsedetection by targeting both M- and S-cones, efficiency of M- and S-coneexcitation should be around 40% and 0.2% correspondingly). Data wereanalyzed using custom code in Matlab, spike sorting was performed usingPlexon Offline Sorting.

After three months, among eyes with intact retina, 3 out of 5demonstrated strong light responses, 1 showed median responses and 1showed minimal responses when tested using MEA recording. In 3 retinaswith strong responses, light responses become detectable in the scotopicrange (from 42 to 112 photons×s⁻¹×μm⁻²) and strong responses wereobserved up to the brightest photopic intensities of 2.00×10⁹photons×s⁻¹×μm⁻². Meanwhile retinas from uninjected contralateral eyeshowed minimal to abolished responses. As expected for rod/cone drivenresponses, after exposures to the brightest stimulation series, scotopicresponses disappeared but responses in the photopic range were notsignificantly affected. Response kinetics and sensitivity were verysimilar to those observed from the 5 WT retinas tested under identicalprotocol (sensitivity of the treated retinas being slightly lowercompared to the most sensitive WT retinas for which dimmest flashesproducing rod-driven responses were in the 8-21 photons×s⁻¹×μm⁻² range).One of the treated retinas tested two months after injectiondemonstrated lower light sensitivity (responses started at the photopicintensity of 1.52×10⁴ photons×s¹×μm²) and responses were mostly goneafter brightest exposures. This may indicates that 3 mos are requiredfor sufficient expression of the targeted protein and generation offunctional rod/cone outer segments. All ganglion cell types identifiablein the WT retinas under full field stimulation (ON-, OFF- andON/OFF-types) were detected in the Lca5−/− treated retinas after spikesorting. As well as WT retinas, treated Lca5−/− retinas were responsiveto 4 Hz flicker stimulation in the intensity range of 3.53×10²-2.00×10⁹photons×s¹×μm². Only one of the 7 control untreated Lca5−/− retinasdemonstrated very weak light responses at the bright photopic stimulilikely indicative of the remaining cone function. All untreated Lca5−/−retinas demonstrated slow melanopsin driven responses at brighterintensities which were absent in the light sensitive treated Lca5−/−retinas.

Two of the treated Lca5−/− retinas had signs of post-injection injuryand did not show light responses despite strong spontaneous firing. Onetreated retina did not show obvious signs of post injection injury butdemonstrated only very weak light responses at the brighter end ofstimulation intensities (8.07×10⁸ photons×s⁻¹×μm⁻² and above) whichmight be indicative of the low expression of the targeted protein and/orof the weak remaining cone function.

Given successful injection and enough post injection time to expresstargeted protein and restore function of rod/cone outer segments, genetherapy restores degenerated retinal cells to a state nearlyindistinguishable from the WT conditions.

In summary, LCA itself is one of the most severe retinal degenerativediseases, and LCA5 is one of the most severe subtypes within thiscategory. Often LCA5 patients enjoy only light perception early in life,and it is difficult even to carry out structural studies and specializedtests of visual function in these ultra-low vision subjects due tonystagmus. Thus, this disease is considered difficult to approach.However, the phenotype of the Lca5−/− mouse reflects many of theclinical findings in humans with LCA5 mutations. The rescue data in theLca5−/− mouse provide hope that a similar gene augmentation approach inhumans could result in improved vision. In parallel with the preclinicalstudies leading to a human clinical trial, it is important to developthe methodology with which to accurately measure the structure andfunction of the LCA5 retina so that the effects of gene therapy in aclinical trial can be captured reliably and accurately. Not only couldsuch studies and a gene therapy clinical trial lead to a treatment forthis devastating condition, but they could also provide the frameworkfor measuring the effects of intervention in other severe, early onsetblinding conditions.

Example 9: Preliminary Analysis of LCA5 Disease in Humans

PLR testing was carried out to determine whether there was any evidenceof function in the residual photoreceptors present in human adult withhomozygous LCA5 mutations. As shown in Figure S11, PLRs were present inthis individual with the same temporal sequence as those in anormal-sighted individual. However, the amplitudes of response werediminished considerably compared to the normal-sighted individual.

Example 10: Loss of Lebercilin Causes Dysregulation of RPE Maturationand Ciliary Function in Cellular and Animal Models

The pathological changes occurring in the visual system in LeberCongenital Amaurosis 5 (LCA5), a disease caused by loss of expression ofthe Lebercilin-encoding LCA5 gene, were explored. Previous studieselucidated the detrimental effect of Lebercilin deficiency on theneuroretina, and specifically on photoreceptors. This study aimed tofurther elucidate the pathogenic mechanisms leading to LCA5 by focusingon the contribution of normal LCA5 expression to the development andfunction of the retinal pigmented epithelium (RPE).

Two independent experimental paradigms were implemented: One usedgeneration of RPE cells from induced pluripotent stem cells (iPSCs) fromboth normal sighted individuals and those with LCA5. RPE cellmorphology, pigmentation, cell-specific markers and characteristics ofthe primary cilia were measured. The second approach evaluated studiesof the retinas of wildtype mice compared to those of a murine model forLCA5 deficiency (LCA5gt/gt mice). The spacial-temporal differentiationpatterns were characterized in order to delineate the degenerative vsdevelopmental changes occurring in the visual system caused by lack ofLCA5 protein.

The results demonstrate that LCA5 deficiency in both human RPE cellmodels and in the RPE of living mice causes profound alterations in thedevelopment of those cells. Through gene expression analysis, weidentified the dysregulation of key proteins responsible forciliogenesis and intraflagellar transport, pigmentation, anddevelopmental WNT signaling pathway. Immunostaining also identifieddifferences in the epithelial barrier consistent with altered maturationdue to loss of LCA5 protein function.

This work reveals the detrimental effect of LCA5 suppression in RPEthrough alteration of processes such as pigmentation, potentially due toan inhibition of intracellular trafficking or indirectly by delaying thematuration progression of these cells. In this study, we introduce thepotential primary role of RPE cells in retinal pathology traditionallyattributed to photoreceptor malfunction.

All patents, patent publications, and other publications cited in thisspecification are incorporated herein by reference, as well as thepriority applications, U.S. Provisional Patent Application No.62/465,649, filed Mar. 1, 2017, and U.S. Provisional Patent ApplicationNo. 62/469,642, filed Mar. 10, 2017. Similarly, the SEQ ID NOs which arereferenced herein and which appear in the appended Sequence Listing areincorporated by reference. While the invention has been described withreference to particular embodiments, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe appended claims.

SEQ Protein or Nucleic ID NO Acid Sequence Description 1 ProteinLebercilin 2 Nucleic Acid native LCA5 3 Nucleic Acid codon optimizedLCA5 4 Nucleic Acid NM_181714.3 (transcript variant 1) 5 Nucleic AcidNM_001122769.2 (transcript variant 2) 6 Nucleic Acid XM_011535504.1(transcript variant X1) 7 Nucleic Acid XM_005248665.4 (transcriptvariant X2) 8 Nucleic Acid production plasmid 9 Nucleic Acid productionplasmid 10 Nucleic Acid promoter 11 Nucleic Acid AAV7m8 capsid 12Protein AAV7m8 capsid 13 Nucleic Acid LCA-13F6 primer 14 Nucleic AcidLCA-13R2 primer 15 Nucleic Acid en2.8r primer

Sequence Listing Free Text

The following information is provided for sequences containing free textunder numeric identifier <223>.

SEQ ID NO: (containing free text) Free text under <223> 3 <223>constructed sequence 8 <223> constructed sequence 9 <223> constructedsequence 10 <223> constructed sequence 11 <223> constructed sequence 12<223> constructed sequence 13 <223> constructed sequence 14 <223>constructed sequence 15 <223> constructed sequence

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1. A nucleic acid sequence encoding human lebercilin, wherein thenucleic acid sequence is at least 90% identical to SEQ ID NO:
 3. 2. Arecombinant adeno-associated virus (rAAV) comprising an AAV capsidprotein and the nucleic acid of claim
 1. 3. The rAAV of claim 2, whereinthe nucleotide sequence is at least 95% identical to SEQ ID NO:
 3. 4.The rAAV of claim 2, wherein the nucleotide sequence is operativelyassociated with expression control sequences that directs expression ofthe nucleotide sequence in a host cell.
 5. An rAAV expression cassettecomprising the rAAV of claim 4, a 5′ AAV inverted terminal repeat (ITR),and a 3′ AAV ITR.
 6. A plasmid comprising the expression cassette ofclaim
 5. 7. The rAAV of claim 4, wherein the host cell is a human cell.8. The rAAV of claim 4, wherein the host cell is a photoreceptor cell.9. The rAAV of claim 4, wherein the expression control sequencescomprise a rhodopsin kinase promoter sequence.
 10. The rAAV of claim 4,wherein the expression control sequences comprise a cytomegalovirus(CMV) promoter sequence or a hybrid promoter sequence comprising a CMVpromoter sequence and a chicken beta actin (CBA) promoter sequence. 11.The rAAV of claim 1, wherein the AAV capsid protein is an AAV8 capsid,or variant thereof, an AAV7 capsid, or variant thereof, an AAV5 capsid,or variant thereof, or an AAV2 capsid or variant thereof.
 12. The rAAVof claim 1, wherein the AAV capsid protein is an AAV8 capsid.
 13. A hostcell comprising the rAAV of claim
 1. 14. A composition comprising therAAV of claim 1 and a carrier or excipient suitable for delivery to aplurality of ocular cells of a subject.
 15. The composition of claim 14,comprised in a lipid delivery vehicle.
 16. The composition of claim 15,wherein the lipid delivery vehicle is a liposome.
 17. A recombinantadeno-associated virus (rAAV) comprising an AAV8 capsid and anexpression cassette comprising: a) a 5′ AAV ITR; b) a rhodopsin kinasepromoter; c) a nucleotide sequence at least 90% identical to SEQ ID NO:3; and d) a 3′AAV ITR.
 18. The rAAV of claim 17, wherein the nucleotidesequence is at least 95% identical to SEQ ID NO:
 3. 19. A compositioncomprising the rAAV of claim 17, and a pharmaceutically acceptableexcipient.
 20. The composition of claim 19, wherein the pharmaceuticallyacceptable excipient is a carrier or excipient suitable for delivery toa plurality of ocular cells of a subject.
 21. The composition of claim19, comprised in a lipid delivery vehicle.
 22. The composition of claim21, wherein the lipid delivery vehicle is a liposome.
 23. Thecomposition of claim 20, wherein the subject is a human.
 24. A method oftreating a subject with an eye disease or disorder, the methodcomprising administering to the subject a recombinant adeno-associatedvirus (rAAV) comprising an AAV capsid protein and a nucleic acidcomprising a nucleotide sequence at least 90% identical to SEQ ID NO: 3.